Compounds specific to adenosine A2a receptor and uses thereof

ABSTRACT

This invention pertains to compounds which specifically inhibit the adenosine A 2a  receptor and the use of these compounds to treat a disease associated with A 2a  adenosine receptor in a subject, comprising administering to the subject a therapeutically effective amount of the compounds.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/169,037, filed Dec. 2, 1999.

[0002] Throughout this application, various publications are referencedby author and date. Full citations for these publications may be foundthroughout the specification. The disclosures of these publications intheir entireties are hereby incorporated by reference into thisapplication in order to more fully describe the state of the art asknown to those skilled therein as of the date of the invention describedand claimed herein.

BACKGROUND OF THE INVENTION

[0003] Adenosine is an ubiquitous modulator of numerous physiologicalactivities, particularly within the cardiovascular and nervous systems.The effects of adenosine appear to be mediated by specific cell surfacereceptor proteins. Adenosine modulates diverse physiological functionsincluding induction of sedation, vasodilation, suppression of cardiacrate and contractility, inhibition of platelet aggregability,stimulation of gluconeogenesis and inhibition of lipolysis. In additionto its effects on adenylate cyclase, adenosine has been shown to openpotassium channels, reduce flux through calcium channels, and inhibit orstimulate phosphoinositide turnover through receptor-mediated mechanisms(See for example, C. E. Muller and B. Stein “Adenosine ReceptorAntagonists: Structures and Potential Therapeutic Applications,” CurrentPharmaceutical Design, 2:501 (1996) and C. E. Muller “A₁-AdenosineReceptor Antagonists,” Exp. Opin. Ther. Patents 7(5):419 (1997)).

[0004] Adenosine receptors belong to the superfamily of purine receptorswhich are currently subdivided into P₁ (adenosine) and P₂ (ATP, ADP, andother nucleotides) receptors. Four receptor subtypes for the nucleosideadenosine have been cloned so far from various species including humans.Two receptor subtypes (A₁ and A_(2a)) exhibit affinity for adenosine inthe nanomolar range while two other known subtypes A_(2b) and A₃ arelow-affinity receptors, with affinity for adenosine in thelow-micromolar range. A₁ and A₃ adenosine receptor activation can leadto an inhibition of adenylate cyclase activity, while A_(2a) and A_(2b)activation causes a stimulation of adenylate cyclase.

[0005] A few A₁ antagonists have been developed for the treatment ofcognitive disease, renal failure, and cardiac arrhythmias. It has beensuggested that A_(2a) antagonists may be beneficial for patientssuffering from Morbus Parkinson (Parkinson's disease). Particularly inview of the potential for local delivery, adenosine receptor antagonistsmay be valuable for treatment of allergic inflammation and asthma.Available information (for example, Nyce & Metzger “DNA antisenseTherapy for Asthma in an Animal Model” Nature (1997) 385:721-5)indicates that in this pathophysiologic context, A₁ antagonistsmay block contraction of smooth muscle underlying respiratory epithelia,while A_(2b) or A₃ receptor antagonists may block mast celldegranulation, mitigating the release of histamine and otherinflammatory mediators. A_(2b) receptors have been discovered throughoutthe gastrointestinal tract, especially in the colon and the intestinalepithelia. It has been suggested that A_(2b) receptors mediate cAMPresponse (Strohmeier et al., J. Bio. Chem. (1995) 270:2387-94).

[0006] Adenosine receptors have also been shown to exist on the retinasof various mammalian species including bovine, porcine, monkey, rat,guinea pig, mouse, rabbit and human (See, Blazynski et al., DiscreteDistributions of Adenosine Receptors in Mammalian Retina, Journal ofNeurochemistry, volume 54, pages 648-65S (1990); Woods et al.,Characterization of Adenosine A ₁-Receptor Binding Sites in BovineRetinal Membranes, Experimental Eye Research, volume 53, pages 325-331(1991); and Braas et al., Endogenous adenosine and adenosine receptorslocalized to ganglion cells of the retina, Proceedings of the NationalAcademy of Science, volume 84, pages 3906-3910 (1987)). Recently,Williams reported the observation of adenosine transport sites in acultured human retinal cell line (Williams et al., Nucleoside TransportSites in a Cultured Human Retinal Cell Line Established By SV-40 TAntigen Gene, Current Eye Research, volume 13, pages 109-118 (1994)).

[0007] Compounds which regulate the uptake of adenosine uptake havepreviously been suggested as potential therapeutic agents for thetreatment of retinal and optic nerve head damage. In U.S. Pat. No.5,780,450 to Shade, Shade discusses the use of adenosine uptakeinhibitors for treating eye disorders. Shade does not disclose the useof specific A₃ receptor inhibitors. The entire contents of U.S. Pat. No.5,780,450 are hereby incorporated herein by reference.

[0008] Additional adenosine receptor antagonists are needed aspharmacological tools and are of considerable interest as drugs for theabove-referenced disease states and/or conditions.

SUMMARY OF THE INVENTION

[0009] The present invention is based on compounds which selectivelybind to adenosine A_(2a) receptor, thereby treating a disease associatedwith A_(2a) adenosine receptor in a subject by administering to thesubject a therapeutically effective amount of such compounds. Thedisease to be treated are associated with, for example, a centralnervous system disorder, a cardiovascular disorder, a renal disorder, aninflammatory disorder, a gastrointestinal disorder, an eye disorder, anallergic disorder or a respiratory disorder.

[0010] The present invention is based, at least in part, on thediscovery that certain N-6 substituted 7-deazapurines, described infra,can be used to treat a N-6 substituted 7-deazapurine responsive state.Examples of such states include those in which the activity of theadenosine receptors is increased, e.g., bronchitis, gastrointestinaldisorders, or asthma. These states can be characterized in thatadenosine receptor activation can lead to the inhibition or stimulationof adenylate cyclase activity. Compositions and methods of the inventioninclude enantiomerically or diastereomerically pure N-6 substituted7-deazapurines. Preferred N-6 substituted 7-deazapurines include thosewhich have an acetamide, carboxamide, substituted cyclohexyl, e.g.,cyclohexanol, or a urea moiety attached to the N-6 nitrogen through analkylene chain.

[0011] The present invention pertains to methods for modulating anadenosine receptor(s) in a mammal by administering to the mammal atherapeutically effective amount of a N-6 substituted 7-deazapurine,such that modulation of the adenosine receptor's activity occurs.Suitable adenosine receptors include the families of A₁, A₂, or A₃. In apreferred embodiment, the N-6 substituted 7-deazapurine is a adenosinereceptor antagonist.

[0012] The invention further pertains to methods for treating N-6substituted 7-deazapurine disorders, e.g., asthma, bronchitis, allergicrhinitis, chronic obstructive pulmonary disease, renal disorders,gastrointestinal disorders, and eye disorders, in a mammal byadministering to the mammal a therapeutically effective amount of a N-6substituted 7-deazapurine, such that treatment of the disorder in themammal occurs. Suitable N-6 substituted 7 deazapurines include thoseillustrated by the general formula I:

[0013] and pharmaceutically acceptable salts thereof. R₁ and R₂ are eachindependently a hydrogen atom or a substituted or unsubstituted alkyl,aryl, or alkylaryl moiety or together form a substituted orunsubstituted heterocyclic ring. R₃ is a substituted or unsubstitutedalkyl, aryl, or alkylaryl moiety. R₄ is a hydrogen atom or a substitutedor unsubstituted alkyl, aryl, or alkylaryl moiety. R₅ and R₆ are eachindependently a halogen atom, e.g., chlorine, fluorine, or bromine, ahydrogen atom or a substituted or unsubstituted alkyl, aryl, oralkylaryl moiety, or R₅ is carboxyl, esters of carboxyl, orcarboxamides, or R₄ and R₅ or R₅ and R₆ together form a substituted orunsubstituted heterocyclic or carbocyclic ring.

[0014] In certain embodiments, R₁ and R₂ can each independently be asubstituted or unsubstituted cycloalkyl or heteroarylalkyl moieties. Inother embodiments, R₃ is a hydrogen atom or a substituted orunsubstituted heteroaryl moiety. In still other embodiments, R₄, R₅ andR₆ can each be independently a heteroaryl moieties. In a preferredembodiment, R₁ is a hydrogen atom, R₂ is a cyclohexanol, e.g.,trans-cyclohexanol, R₃ is phenyl, R₄ is a hydrogen atom, R₅ is a methylgroup and R₆ is a methyl group. In still another embodiment, R₁ is ahydrogen atom, R₂ is

[0015] R₃ is phenyl, R₄ is a hydrogen atom and R₅ and R₆ are methylgroups.

[0016] The invention further pertains to pharmaceutical compositions fortreating a N-6 substituted 7-deazapurine responsive state in a mammal,e.g., asthma, bronchitis, allergic rhinitis, chronic obstructivepulmonary disease, renal disorders, gastrointestinal disorders, and eyedisorders. The pharmaceutical composition includes a therapeuticallyeffective amount of a N-6 substituted 7-deazapurine and apharmaceutically acceptable carrier.

[0017] The present invention also pertains to packaged pharmaceuticalcompositions for treating a N-6 substituted 7-deazapurine responsivestate in a mammal. The packaged pharmaceutical composition includes acontainer holding a therapeutically effective amount of at least one N-6substituted 7-deazapurine and instructions for using the N-6 substituted7-deazapurine for treating a N-6 substituted 7-deazapurine responsivestate in a mammal.

[0018] The invention further pertains to compounds of formula I wherein

[0019] R₁ is hydrogen;

[0020] R₂ is substituted or unsubstituted cycloalkyl, substituted orunsubstituted alkyl, or R₁ and R₂ together form a substituted orunsubstituted heterocyclic ring;

[0021] R₃ is unsubstituted or substituted aryl;

[0022] R₄ is hydrogen; and

[0023] R₅ and R₆ are each independently hydrogen or alkyl, andpharmaceutically acceptable salts thereof. The deazapurines of thisembodiment may advantageously be selective A_(2a) receptor antagonists.These compounds may be useful for numerous therapeutic uses such as, forexample, the treatment of asthma, kidney failure associated with heartfailure, and glaucoma. In a particularly preferred embodiment, thedeazapurine is a water soluble prodrug that is capable of beingmetabolized in vivo to an active drug by, for example, esterasecatalyzed hydrolysis.

[0024] In yet another embodiment, the invention features a method forinhibiting the activity of an adenosine receptor (e.g., A_(2a)) in acell, by contacting the cell with N-6 substituted 7-deazapurine (e.g.,preferably, an adenosine receptor antagonist).

[0025] In another aspect, the invention features a method for treatingdamage to the eye of an animal(e.g., a human) by administering to theanimal an effective amount of an N-6 substituted 7-deazapurine offormula I. Preferably, the N-6 substituted 7-deazapurine is anantagonist of A_(2a) adenosine receptors in cells of the animal. Thedamage is to the retina or the optic nerve head and may be acute orchronic. The damage may be the result of, for example, glaucoma, edema,ischemia, hypoxia or trauma.

[0026] The invention also features a pharmaceutical compositioncomprising a N-6 substituted compound of formula I. Preferably, thepharmaceutical preparation is an ophthalmic formulation (e.g., anperiocular, retrobulbar or intraocular injection formulation, a systemicformulation, or a surgical irrigating solution).

[0027] In yet another embodiment, the invention features a compoundhaving the formula II:

[0028] wherein X is N or CR₆; R₁ and R₂ are each independently hydrogen,or substituted or unsubstituted alkoxy, aminoalkyl, alkyl, aryl, oralkylaryl, or together form a substituted or unsubstituted heterocyclicring, provided that both R₁ and R₂ are both not hydrogen; R₃ issubstituted or unsubstituted alkyl, arylalkyl, or aryl; R₄ is hydrogenor substituted or unsubstituted C₁-C₆ alkyl; L is hydrogen, substitutedor unsubstituted alkyl, or R₄ and L together form a substituted orunsubstituted heterocyclic or carbocyclic ring; R₆ is hydrogen,substituted or unsubstituted alkyl, or halogen; Q is CH₂, O, S, or NR₇,wherein R₇ is hydrogen or substituted or unsubstituted C₁-C₆ alkyl; andW is unsubstituted or substituted alkyl, cycloalkyl, aryl, arylalkyl,biaryl, heteroaryl, substituted carbonyl, substituted thiocarbonyl, orsubstituted sulfonyl; provided that if R₃ is pyrrolidino, then R₄ is notmethyl. The invention also pertains to pharmaceutically acceptable saltsand prodrugs of the compounds of the invention.

[0029] In an advantageous embodiment, X is CR₆ and Q is CH₂, O, S, or NHin formula II, wherein R₆ is as defined above.

[0030] In another embodiment of formula II, X is N.

[0031] The invention further pertains to a method for inhibiting theactivity of an adenosine receptor (e.g., an A_(2b) adenosine receptor)in a cell by contacting the cell with a compound of the invention.Preferably, the compound is an antagonist of the receptor.

[0032] The invention also pertains to a method for treating agastrointestinal disorder (e.g., diarrhea) or a respiratory disorder(e.g., allergic rhinitis, chronic obstructive pulmonary disease) in ananimal by administering to an animal an effective amount of a compoundof formula II (e.g., an antagonist of A_(2b)). Preferably, the animal isa human.

[0033] This invention also features a compound having the structure:

[0034] wherein NR₁R₂ is a substituted or unsubstituted 4-8 memberedring;

[0035] wherein Ar is a substituted or unsubstituted four to six memberedring;

[0036] wherein R₄ is H, alkyl, substituted alkyl, aryl, arylalkyl,amino, substituted aryl, wherein said substituted alkyl is —C(R₈)(R₉)XR₆, wherein X is O, S, or NR₇, wherein R₈ and R₉ are eachindependently H or alkyl, wherein R₆ and R₇ are each independently alkylor cycloalkyl, or R₆, R₇ and the nitrogen together form a substituted orunsubstituted ring of between 4 and 7 members.

[0037] wherein R₅ is wherein R₅ is H, alkyl, substituted alkyl, orcycloalkyl;

[0038] with the proviso that NR₁R₂ is not 3-acetamido piperadino,3-hydroxy pyrrolidino, 3-methyloxy carbonylmethyl pyrrolidino,3-aminocarbonylmethyl, or pyrrolidino; with the proviso that NR₁R₂ is3-hydroxymethyl piperadino only when Ar is 4-pyridyl.

[0039] This invention also features a method for inhibiting the activityof an A_(2a) adenosine receptor in a cell, which comprises contactingsaid cell with the above-mentioned compounds.

DETAILED DESCRIPTION

[0040] The features and other details of the invention will now be moreparticularly described and pointed out in the claims. It will beunderstood that the particular embodiments of the invention are shown byway of illustration and not as limitations of the invention. Theprinciple features of this invention can be employed in variousembodiments without departing from the scope of the invention.

[0041] The present invention pertains to methods for treating a N-6substituted 7-deazapurine responsive state in a mammal. The methodsinclude administration of a therapeutically effective amount of a N-6substituted 7-deazapurine, described infra, to the mammal, such thattreatment of the N-6 substituted 7-deazapurine responsive state in themammal occurs.

[0042] The language “N-6 substituted 7-deazapurine responsive state” isintended to include a disease state or condition characterized by itsresponsiveness to treatment with a N-6 substituted 7-deazapurine of theinvention as described infra, e.g., the treatment includes a significantdiminishment of at least one symptom or effect of the state achievedwith a N-6 substituted 7-deazapurine of the invention. Typically suchstates are associated with an increase of adenosine within a host suchthat the host often experiences physiological symptoms which include,but are not limited to, release of toxins, inflammation, coma, waterretention, weight gain or weight loss, pancreatitis, emphysema,rheumatoid arthritis, osteoarthritis, multiple organ failure, infant andadult respiratory distress syndrome, allergic rhinitis, chronicobstructive pulmonary disease, eye disorders, gastrointestinaldisorders, skin tumor promotion, immunodeficiency and asthma. (See forexample, C. E. Muller and B. Stein “Adenosine Receptor Antagonists:Structures and Potential Therapeutic Applications,” CurrentPharmaceutical Design, 2:501 (1996) and C. E. Muller “A₁-AdenosineReceptor Antagonists,” Exp. Opin. Ther. Patents 7 (5):419 (1997) and I.Feoktistove, R. Polosa, S. T. Holgate and I. Biaggioni “Adenosine A_(2B)receptors: a novel therapeutic target in asthma?” TiPS 19; 148 (1998)).The effects often associated with such symptoms include, but are notlimited to, fever, shortness of breath, nausea, diarrhea, weakness,headache, and even death. In one embodiment, a N-6 substituted7-deazapurine responsive state includes those disease states which aremediated by stimulation of adenosine receptors, e.g., A₁, A_(2a),A_(2b), A₃, etc., such that calcium concentrations in cells and/oractivation of PLC (phospholipase C) is modulated. In a preferredembodiment, a N-6 substituted 7-deazapurine responsive state isassociated with adenosine receptor(s) e.g., the N-6 substituted7-deazapurine acts as an antagonist. Examples of suitable responsivestates which can be treated by the compounds of the invention, e.g.,adenosine receptor subtypes which mediate biological effects, includecentral nervous system (CNS) effects, cardiovascular effects, renaleffects, respiratory effects, immunological effects, gastrointestinaleffects and metabolic effects. The relative amount of adenosine in asubject can be associated with the effects listed below; that isincreased levels of adenosine can trigger an effect, e.g., an undesiredphysiological response, e.g., an asthmatic attack.

[0043] CNS effects include decreased transmitter release (A₁), sedation(A₁), decreased locomotor activity (A_(2a)), anticonvulsant activity,chemoreceptor stimulation (A₂) and hyperalgesia. Therapeuticapplications of the inventive compounds include treatment of dementia,Alzheimer's disease and memory enhancement.

[0044] Cardiovascular effects include vasodilation (A_(2a)), (A_(2b))and (A₃), vasoconstriction (A₁), bradycardia (A₁), platelet inhibition(A_(2a)), negative cardiac inotropy and dromotropy (A₁), arrhythmia,tachycardia and angiogenesis. Therapeutic applications of the inventivecompounds include, for example, prevention of ischaemia-inducedimpairment of the heart and cardiotonics, myocardial tissue protectionand restoration of cardiac function.

[0045] Renal effects include decreased GFR (A₁), mesangial cellcontraction (A₁), antidiuresis (A₁) and inhibition of renin release(A₁). Suitable therapeutic applications of the inventive compoundsinclude use of the inventive compounds as diuretic, natriuretic,potassium-sparing, kidney-protective/prevention of acute renal failure,antihypertensive, anti-oedematous and anti-nephritic agents.

[0046] Respiratory effects include bronchodilation (A₂),bronchoconstriction (A₁), chronic obstructive pulmonary disease,allergic rhinitis, mucus secretion and respiratory depression (A₂).Suitable therapeutic applications for the compounds of the inventioninclude anti-asthmatic applications, treatment of lung disease aftertransplantation and respiratory disorders.

[0047] Immunological effects include immunosuppression (A₂), neutrophilchemotaxis (A₁), neutrophil superoxide generation (A_(2a)) and mast celldegranulation (A_(2b) and A₃) Therapeutic applications of antagonistsinclude allergic and non allergic inflammation, e.g., release ofhistamine and other inflammatory mediators.

[0048] Gastrointestinal effects include inhibition of acid secretion(A₁) therapeutic application may include reflux and ulcerativeconditions Gastrointestinal effects also include colonic, intestinal anddiarrheal disease, e.g., diarrheal disease associated with intestinalinflammation (A_(2b)).

[0049] Eye disorders include retinal and optic nerve head injury andtrauma related disorders (A₃). In a preferred embodiment, the eyedisorder is glaucoma.

[0050] Other therapeutic applications of the compounds of the inventioninclude treatment of obesity (lipolytic properties), hypertension,treatment of depression, sedative, anxiolytic, as antileptics and aslaxatives, e.g., effecting motility without causing diarrhea.

[0051] The term “disease state” is intended to include those conditionscaused by or associated with unwanted levels of adenosine, adenylylcyclase activity, increased physiological activity associated withaberrant stimulation of adenosine receptors and/or an increase in cAMP.In one embodiment, the disease state is, for example, asthma, chronicobstructive pulmonary disease, allergic rhinitis, bronchitis, renaldisorders, gastrointestinal disorders, or eye disorders. Additionalexamples include chronic bronchitis and cystic fibrosis. Suitableexamples of inflammatory diseases include non-lymphocytic leukemia,myocardial ischaemia, angina, infarction, cerebrovascular ischaemia,intermittent claudication, critical limb ischemia, venous hypertension,varicose veins, venous ulceration and arteriosclerosis. Impairedreperfusion states include, for example, any post-surgical trauma, suchas reconstructive surgery, thrombolysis or angioplasty.

[0052] The language “treatment of a N-6 substituted 7-deazapurineresponsive state” or “treating a N-6 substituted 7-deazapurineresponsive state” is intended to include changes in a disease state orcondition, as described above, such that physiological symptoms in amammal can be significantly diminished or minimized. The language alsoincludes control, prevention or inhibition of physiological symptoms oreffects associated with an aberrant amount of adenosine. In onepreferred embodiment, the control of the disease state or condition issuch that the disease state or condition is eradicated. In anotherpreferred embodiment, the control is selective such that aberrant levelsof adenosine receptor activity are controlled while other physiologicsystems and parameters are unaffected.

[0053] The term “N-6 substituted 7-deazapurine” is art recognized and isintended to include those compounds having the formula I:

[0054] “N-substituted 7-deazapurine” includes pharmaceuticallyacceptable salts thereof, and, in one embodiment, also includes certainN-6 substituted purines described herein.

[0055] In certain embodiments, the N-6 substituted 7-deazapurine is notN-6 benzyl or N-6 phenylethyl substituted. In other embodiments, R₄ isnot benzyl or phenylethyl substituted. In preferred embodiments, R, andR₂ are both not hydrogen atoms. In still other preferred embodiments, R₃is not a hydrogen atom.

[0056] The language “therapeutically effective amount” of an N-6substituted 7-deazapurine, described infra, is that amount of atherapeutic compound necessary or sufficient to perform its intendedfunction within a mammal, e.g., treat a N-6 substituted 7-deazapurineresponsive state, or a disease state in a mammal. An effective amount ofthe therapeutic compound can vary according to factors such as theamount of the causative agent already present in the mammal, the age,sex, and weight of the mammal, and the ability of the therapeuticcompounds of the present invention to affect a N-6 substituted7-deazapurine responsive state in the mammal.

[0057] One of ordinary skill in the art would be able to study theaforementioned factors and make a determination regarding the effectiveamount of the therapeutic compound without undue experimentation. An invitro or in vivo assay also can be used to determine an “effectiveamount” of the therapeutic compounds described infra. The ordinarilyskilled artisan would select an appropriate amount of the therapeuticcompound for use in the aforementioned assay or as a therapeutictreatment.

[0058] A therapeutically effective amount preferably diminishes at leastone symptom or effect associated with the N-6 substituted 7-deazapurineresponsive state or condition being treated by at least about 20%, (morepreferably by at least about 40%, even more preferably by at least about60%, and still more preferably by at least about 80%) relative tountreated subjects. Assays can be designed by one skilled in the art tomeasure the diminishment of such symptoms and/or effects. Any artrecognized assay capable of measuring such parameters are intended to beincluded as part of this invention. For example, if asthma is the statebeing treated, then the volume of air expended from the lungs of asubject can be measured before and after treatment for measurement ofincrease in the volume using an art recognized technique. Likewise, ifinflammation is the state being treated, then the area which is inflamedcan be measured before and after treatment for measurement ofdiminishment in the area inflamed using an art recognized technique.

[0059] The term “cell” includes both prokaryotic and eukaryotic cells.

[0060] The term “animal” includes any organism with adenosine receptorsor any organism susceptible to a N-6-substituted 7-deazapurineresponsive state. Examples of animals include yeast, mammals, reptiles,and birds. It also includes transgenic animals.

[0061] The term “mammal” is art recognized and is intended to include ananimal, more preferably a warm-blooded animal, most preferably cattle,sheep, pigs, horses, dogs, cats, rats, mice, and humans. Mammalssusceptible to a N-6 substituted 7-deazapurine responsive state,inflammation, emphysema, asthma, central nervous system conditions, oracute respiratory distress syndrome, for example, are included as partof this invention.

[0062] In another aspect, the present invention pertains to methods formodulating an adenosine receptor(s) in a mammal by administering to themammal a therapeutically effective amount of a N-6 substituted7-deazapurine, such that modulation of the adenosine receptor in themammal occurs. Suitable adenosine receptors include the families of A₁,A₂, or A₃. In a preferred embodiment, the N-6 substituted 7-deazapurineis an adenosine receptor antagonist.

[0063] The language “modulating an adenosine receptor” is intended toinclude those instances where a compound interacts with an adenosinereceptor(s), causing increased, decreased or abnormal physiologicalactivity associated with an adenosine receptor or subsequent cascadeeffects resulting from the modulation of the adenosine receptor.Physiological activities associated with adenosine receptors includeinduction of sedation, vasodilation, suppression of cardiac rate andcontractility, inhibition of platelet aggregability, stimulation ofgluconeogenesis, inhibition of lipolysis, opening of potassium channels,reducing flux of calcium channels, etc.

[0064] The terms “modulate”, “modulating” and “modulation” are intendedto include preventing, eradicating, or inhibiting the resulting increaseof undesired physiological activity associated with abnormal stimulationof an adenosine receptor, e.g., in the context of the therapeuticmethods of the invention. In another embodiment, the term modulateincludes antagonistic effects, e.g., diminishment of the activity orproduction of mediators of allergy and allergic inflammation whichresults from the overstimulation of adenosine receptor(s). For example,the therapeutic deazapurines of the invention can interact with anadenosine receptor to inhibit, for example, adenylate cyclase activity.

[0065] The language “condition characterized by aberrant adenosinereceptor activity” is intended to include those diseases, disorders orconditions which are associated with aberrant stimulation of anadenosine receptor, in that the stimulation of the receptor causes abiochemical and or physiological chain of events that is directly orindirectly associated with the disease, disorder or condition. Thisstimulation of an adenosine receptor does not have to be the solecausative agent of the disease, disorder or condition but merely beresponsible for causing some of the symptoms typically associated withthe disease, disorder, or condition being treated. The aberrantstimulation of the receptor can be the sole factor or at least one otheragent can be involved in the state being treated. Examples of conditionsinclude those disease states listed supra, including inflammation,gastrointestinal disorders and those symptoms manifested by the presenceof increased adenosine receptor activity. Preferred examples includethose symptoms associated with asthma, allergic rhinitis, chronicobstructive pulmonary disease, emphysema, bronchitis, gastrointestinaldisorders and glaucoma.

[0066] The language “treating or treatment of a condition characterizedby aberrant adenosine receptor activity” is intended to include thealleviation of or diminishment of at least one symptom typicallyassociated with the condition. The treatment also includes alleviationor diminishment of more than one symptom. Preferably, the treatmentcures, e.g., substantially eliminates, the symptoms associated with thecondition.

[0067] The present invention pertains to compounds, N-6 substituted7-deazapurines, having the formula I:

[0068] wherein R₁ and R₂ are each independently a hydrogen atom or asubstituted or unsubstituted alkyl, aryl, or alkylaryl moiety ortogether form a substituted or unsubstituted heterocyclic ring; R₃ is ahydrogen atom or a substituted or unsubstituted alkyl, aryl, oralkylaryl moiety; R₄ is a hydrogen atom or a substituted orunsubstituted alkyl, aryl, or alkylaryl moiety. R₅ and R₆ are eachindependently a halogen atom, e.g., chlorine, fluorine, or bromine, ahydrogen atom or a substituted or unsubstituted alkyl, aryl, oralkylaryl moiety or R₄ and R₅ or R₅ and R₆ together form a substitutedor unsubstituted heterocyclic or carbocyclic ring. Also included, arepharmaceutically acceptable salts of the N-6 substituted 7-deazapurines.

[0069] In certain embodiments, R₁ and R₂ can each independently be asubstituted or unsubstituted cycloalkyl or heteroarylalkyl moieties. Inother embodiments, R₃ is a hydrogen atom or a substituted orunsubstituted heteroaryl moiety. In still other embodiments, R₄, R₅ andR₆ can each be independently a heteroaryl moiety.

[0070] In one embodiment, R₁ is a hydrogen atom, R₂ is a substituted orunsubstituted cyclohexane, cyclopentyl, cyclobutyl or cyclopropanemoiety, R₃ is a substituted or unsubstituted phenyl moiety, R₄ is ahydrogen atom and R₅ and R₆ are both methyl groups.

[0071] In another embodiment, R₂ is a cyclohexanol, a cyclohexanediol, acyclohexylsulfonamide, a cyclohexanamide, a cyclohexylester, acyclohexene, a cyclopentanol or a cyclopentanediol and R₃ is a phenylmoiety.

[0072] In still another embodiment, R₁ is a hydrogen atom, R₂ is acyclohexanol, R₃ is a substituted or unsubstituted phenyl, pyridine,furan, cyclopentane, or thiophene moiety, R₄ is a hydrogen atom, asubstituted alkyl, aryl or arylalkyl moiety, and R₅ and R₆ are eachindependently a hydrogen atom, or a substituted or unsubstituted alkyl,aryl, or alkylaryl moiety.

[0073] In yet another embodiment, R₁ is a hydrogen atom, R₂ issubstituted or unsubstituted alkylamine, arylamine, or alkylarylamine, asubstituted or unsubstituted alkylamide, arylamide or alkylarylamide, asubstituted or unsubstituted alkylsulfonamide, arylsulfonamide oralkylarylsulfonamide, a substituted or unsubstituted alkylurea, arylureaor alkylarylurea, a substituted or unsubstituted alkylcarbamate,arylcarbamate or alkylarylcarbamate, a substituted or unsubstitutedalkylcarboxylic acid, arylcarboxylic acid or alkylarylcarboxylic acid,R₃ is a substituted or unsubstituted phenyl moiety, R₄ is a hydrogenatom and R₅ and R₆ are methyl groups.

[0074] In still another embodiment, R₂ is guanidine, a modifiedguanidine, cyanoguanidine, a thiourea, a thioamide or an amidine.

[0075] In one embodiment, R₂ can be

[0076] wherein R_(2a)-R_(2c) are each independently a hydrogen atom or asaturated or unsaturated alkyl, aryl or alkylaryl moiety and R_(2d) is ahydrogen atom or a saturated or unsaturated alkyl, aryl, or alkylarylmoiety, NR_(2e)R_(2f), or OR_(2g), wherein R_(2e)-R_(2g) are eachindependently a hydrogen atom or a saturated or unsaturated alkyl, arylor alkylaryl moieties. Alternatively, R_(1a) and R_(2b) together canform a carbocyclic or heterocyclic ring having a ring size between about3 and 8 members, e.g., cyclopropyl, cyclopentyl, cyclohexyl groups.

[0077] In one aspect of the invention, both R₅ and R₆ are not methylgroups, preferably, one of R₅ and R₆ is an alkyl group, e.g., a methylgroup, and the other is a hydrogen atom.

[0078] In another aspect of the invention, when R₄ is 1-phenylethyl andR₁ is a hydrogen atom, then R₃ is not phenyl, 2-chlorophenyl,3-chlorophenyl, 4-chlorophenyl, 3,4-dichlorophenyl, 3-methoxyphenyl or4-methoxyphenyl or when R₄ and R₁ are 1-phenylethyl, then R₃ is not ahydrogen atom or when R₄ is a hydrogen atom and R₃ is a phenyl, then R₁is not phenylethyl.

[0079] In another aspect of the invention, when R₅ and R₆ together forma carbocyclic ring, e.g.,

[0080] or pyrimido[4,5-6]indole, then R₃ is not phenyl when R₄ is1-(4-methylphenyl)ethyl, phenylisopropyl, phenyl or 1-phenylethyl orwhen R₃ is not a hydrogen atom when R₄ is 1-phenylethyl. The carbocyclicring formed by R₅ and R₆ can be either aromatic or aliphatic and canhave between 4 and 12 carbon atoms, e.g., naphthyl, phenylcyclohexyl,etc., preferably between 5 and 7 carbon atoms, e.g., cyclopentyl orcyclohexyl. Alternatively, R₅ and R₆ together can form a heterocyclicring, such as those disclosed below. Typical heterocyclic rings includebetween 4 and 12 carbon atoms, preferably between 5 and 7 carbon atoms,and can be either aromatic or aliphatic. The heterocyclic ring can befurther substituted, including substitution of one or more carbon atomsof the ring structure with one or more heteroatoms.

[0081] In still another aspect of the invention, R₁ and R₂ form aheterocyclic ring. Representative examples include, but are not limitedto, those heterocyclic rings listed below, such as morpholino,piperazine and the like, e.g., 4-hydroxypiperidines, 4-aminopiperidines.Where R₁ and R₂ together form a piperazino group,

[0082] wherein R₇ can be a hydrogen atom or a substituted orunsubstituted alkyl, aryl or alkylaryl moiety.

[0083] In yet another aspect of the invention R₄ and R₅ together canform a heterocyclic ring, e.g.,

[0084] wherein the heterocyclic ring can be either aromatic or aliphaticand can form a ring having between 4 and 12 carbon atoms, e.g.,naphthyl, phenylcyclohexyl, etc. and can be either aromatic oraliphatic, e.g., cyclohexyl, cyclopentyl.

[0085] The heterocyclic ring can be further substituted, includingsubstitution of carbon atoms of the ring structure with one or moreheteroatoms. Alternatively, R₄ and R₅ together can form a heterocyclicring, such as those disclosed below.

[0086] In certain embodiments, the N-6 substituted 7-deazapurine is notN-6 benzyl or N-6 phenylethyl substituted. In other embodiments, R₄ isnot benzyl or phenylethyl substituted. In preferred embodiments, R₁ andR₂ are both not hydrogen atoms. In still other preferred embodiments, R₃is not H.

[0087] The compounds of the invention may comprise water-solubleprodrugs which are described in WO 99/33815, International ApplicationNo. PCT/US98/04595, filed Mar. 9, 1998 and published Jul. 8, 1999. Theentire content of WO 99/33815 is expressly incorporated herein byreference. The water-soluble prodrugs are metabolized in vivo to anactive drug, e.g., by esterase catalyzed hydrolysis. Examples ofpotential prodrugs include deazapurines with, for example, R₂ ascycloalkyl substituted with —OC(O) (Z)NH₂, wherein Z is a side chain ofa naturally or unnaturally occurring amino acid, or analog thereof, anα, β, γ, or ω amino acids, or a dipeptide. Preferred amino acid sidechains include those of glycine, alanine, valine, leucine, isoleucine,lysine, α-methylalanine, aminocyclopropane carboxylic acid,azetidine-2-carboxylic acid, β-alanine, γ-aminobutyric acid,alanine-alanine, or glycine-alanine.

[0088] In a further embodiment, the invention features deazapurines ofthe formula (I), wherein R₁ is hydrogen; R₂ is substituted orunsubstituted cycloalkyl, substituted or unsubstituted alkyl, or R₁ andR₂ together form a substituted or unsubstituted heterocyclic ring; R₃ isunsubstituted or substituted aryl; R₄ is hydrogen; and R₅ and R₆ areeach independently hydrogen or alkyl, and pharmaceutically acceptablesalts thereof. The deazapurines of this embodiment may potentially beselective A₃ receptor antagonists.

[0089] In one embodiment, R₂ is substituted (e.g., hydroxy substituted)or unsubstituted cycloalkyl. In an advantageous subembodiment, R₁ and R₄are hydrogen, R₃ is unsubstituted or substituted phenyl, and R₅ and R₆are each alkyl. Preferably R₂ is mono-hydroxycyclopentyl ormono-hydroxycyclohexyl. R₂ also may be substituted with —NH—C(═O)E,wherein E is substituted or unsubstituted C₁-C₄ alkyl (e.g., alkylamine,e.g., ethylamine.).

[0090] R₁ and R₂ may also together form a substituted or unsubstitutedheterocyclic ring, which may be substituted with an amine or acetamidogroup.

[0091] In another aspect, R₂ may be —A—NHC(═O)B, wherein A isunsubstituted C₁-C₄ alkyl (e.g., ethyl, propyl, butyl), and B issubstituted or unsubstituted C₁-C₄ alkyl (e.g., methyl, aminoalkyl,e.g., aminomethyl or aminoethyl, alkylamino, e.g., methylamino,ethylamino), preferably when R₁ and R₄ are hydrogen, R₃ is unsubstitutedor substituted phenyl, and R₅ and R₆ are each alkyl. B may besubstituted or unsubstituted cycloalkyl, e.g., cyclopropyl or1-amino-cyclopropyl.

[0092] In another embodiment, R₃ may be substituted or unsubstitutedphenyl, preferably when R₅ and R₆ are each alkyl. Preferably, R₃ mayhave one or more substituents (e.g., o-, m- or p-chlorophenyl, o-, m- orp- fluorophenyl).

[0093] Advantageously, R₃ may be substituted or unsubstitutedheteroaryl, preferably when R₅ and R₆ are each alkyl. Examples ofheteroaryl groups include pyridyl, pyrimidyl, pyridazinyl, pyrazinyl,pyrrolyl, triazolyl, thioazolyl, oxazolyl, oxadiazolyl, furanyl,methylenedioxyphenyl and thiophenyl. Preferably, R₃ is 2-pyridyl,3-pyridyl, 4-pyridyl, 2-pyrimidyl or 3- pyrimidyl.

[0094] Preferably in one embodiment, R₅ and R₆ are each hydrogen. Inanother, R₅ and R₆ are each methyl.

[0095] In a particularly preferred embodiment, the deazapurines of theinvention are water-soluble prodrugs that can be metabolized in vivo toan active drug, e.g. by esterase catalyzed hydrolysis. Preferably theprodrug comprises an R₂ group which is cycloalkyl substituted with—OC(O) (Z)NH₂, wherein Z is a side chain of a naturally or unnaturallyoccurring amino acid, an analog thereof, an α, β, Γ, or ω amino acid, ora dipeptide. Examples of preferred side chains include the side chainsof glycine, alanine, valine, leucine, isoleucine, lysine,α-methylalanine, aminocyclopropane carboxylic acid,azetidine-2-carboxylic acid, β-alanine, γ-aminobutyric acid,alanine-alanine, or glycine-alanine.

[0096] In a particularly preferred embodiment, Z is a side chain ofglycine, R₂ is cyclohexyl, R₃ is phenyl, and R₅ and R₆ are methyl.

[0097] In another embodiment, the deazapurine is4-(cis-3-hydroxycyclopentyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.

[0098] In another embodiment, the deazapurine is4-(cis-3-(2-aminoacetoxy)cyclopentyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d] pyrimidinetrifluoroacetic acid salt.

[0099] In another embodiment, the deazapurine is 4-(3-acetamido)piperidinyl-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d]pyrimidine.

[0100] In another embodiment, the deazapurine is4-(2-N′-methylureapropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.

[0101] In another embodiment, the deazapurine is4-(2-acetamidobutyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.

[0102] In another embodiment, the deazapurine is4-(2-N′-methylureabutyl) amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.

[0103] In another embodiment, the deazapurine is4-(2-aminocyclopropylacetamidoethyl)amino-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.

[0104] In another embodiment, the deazapurine is4-(trans-4-hydroxycyclohexyl) amino-2-(3-chlorophenyl)-7H-pyrrolo[2,3d]pyrimidine.

[0105] In another embodiment, the deazapurine is4-(trans-4-hydroxycyclohexyl) amino -2-(3-fluorophenyl)-7H-pyrrolo [2,3d]pyrimidine.

[0106] In another embodiment, the deazapurine is4-(trans-4-hydroxycyclohexyl)amino-2-(4-pyridyl)-7H-pyrrolo[2,3d]pyrimidine.

[0107] In yet another embodiment, the invention features a method forinhibiting the activity of an adenosine receptor (e.g., A₁, A_(2A),A_(2B), or, preferably, A₃) in a cell, by contacting the cell with N-6substituted 7-deazapurine (e.g., preferably, an adenosine receptorantagonist)

[0108] In another aspect, the invention features a method for treatingdamage to the eye of an animal(e.g., a human) by administering to theanimal an effective amount of an N-6 substituted 7-deazapurine.Preferably, the N-6 substituted 7-deazapurine is an antagonist of A_(2a)adenosine receptors in cells of the animal. The damage is to the retinaor the optic nerve head and may be acute or chronic. The damage may bethe result of, for example, glaucoma, edema, ischemia, hypoxia ortrauma.

[0109] In a preferred embodiment, the invention features a deazapurinehaving the formula II, supra, wherein X is N or CR₆; R₁ and R₂ are eachindependently hydrogen, or substituted or unsubstituted alkoxy,aminoalkyl, alkyl, aryl, or alkylaryl, or together form a substituted orunsubstituted heterocyclic ring, provided that both R₁ and R₂ are bothnot hydrogen; R₃ is substituted or unsubstituted alkyl, arylalkyl, oraryl; R₄ is hydrogen or substituted or unsubstituted C₁-C₆ alkyl; L ishydrogen, substituted or unsubstituted alkyl, or R₄ and L together forma substituted or unsubstituted heterocyclic or carbocyclic ring; R₆ ishydrogen, substituted or unsubstituted alkyl, or halogen; Q is CH₂, O,S, or NR₇, wherein R₇ is hydrogen or substituted or unsubstituted C₁-C₆alkyl; and W is unsubstituted or substituted alkyl, cycloalkyl, alkynyl,aryl, arylalkyl, biaryl, heteroaryl, substituted carbonyl, substitutedthiocarbonyl, or substituted sulfonyl, provided that if R₃ ispyrrolidino, then R₄ is not methyl.

[0110] In one embodiment, in compounds of formula II, X is CR, and Q isCH₂, O, S, or NH. In another embodiment, X is N.

[0111] In a further embodiment of compounds of formula II, W issubstituted or unsubstituted aryl, 5- or 6-member heteroaryl, or biaryl.W may be substituted with one or more substituents. Examples ofsubstituents include: halogen, hydroxy, alkoxy, amino, aminoalkyl,aminocarboxyamide, CN, CF₃, CO₂RB, CONHR₈, CONR₈R₉, SOR₈, SO₂R₈, andSC₂NR₈R₉, wherein R₈ and R₉ are each independently hydrogen, orsubstituted or unsubstituted alkyl, cycloalkyl, aryl, or arylalkyl.Preferably, W may be substituted or unsubstituted phenyl, e.g.,methylenedioxyphenyl. W also may be a substituted or unsubstituted5-membered heteroaryl ring, e.g., pyrrole, pyrazole, oxazole, imidazole,triazole, tetrazole, furan, thiophene, thiazole, and oxadiazole.Preferably, W may be a 6-member heteroaryl ring, e.g., pyridyl,pyrimidyl, pyridazinyl, pyrazinal, and thiophenyl. In a preferredembodiment, W is 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, or 5-pyrimidyl.

[0112] In one advantageous embodiment of compounds of formula II, Q isNH and W is a 3-pyrazolo ring which is unsubstituted or N-substituted bysubstituted or unsubstituted alkyl, cycloalkyl, aryl, or arylalkyl.

[0113] In another embodiment of compounds of formula II, Q is oxygen,and W is a 2-thiazolo ring which is unsubstituted or substituted bysubstituted or unsubstituted alkyl, cycloalkyl, aryl, or arylalkyl.

[0114] In another embodiment of compounds of formula II, W issubstituted or unsubstituted alkyl, cycloalkyl e.g., cyclopentyl, orarylalkyl. Examples of substituents include halogen, hydroxy ,substituted or unsubstituted alkyl, cycloalkyl, aryl, arylalkyl, orNHR₁₀, wherein R₁₀ is hydrogen, or substituted or unsubstituted alkyl,cycloalkyl, aryl, or arylalkyl.

[0115] In yet another embodiment, the invention features a deazapurineof formula II wherein W is —(CH₂)_(a)—C(═O)Y or —(CH₂)_(a)—C(═S)Y, and ais an integer from 0 to 3, Y is aryl, alkyl, arylalkyl, cycloalkyl,heteroaryl, alkynyl, NHR₁₁R₁₂, or, provided that Q is NH, OR₁₃, whereinR₁₁, R₁₂ and R₁₃ are each independently hydrogen, or unsubstituted orsubstituted alkyl, aryl, arylalkyl, or cycloalkyl. Preferably, Y is a 5-or 6-member heteroaryl ring.

[0116] Furthermore, W may be —(CH₂)_(b)—S(═O)_(j)Y, wherein j is 1 or 2,b is 0, 1, 2, or 3, Y is aryl, alkyl, arylalkyl, cycloalkyl, alkynyl,heteroaryl, NHR₁₄R₁₅, provided that when b is 1, Q is CH₂, and whereinR₁₄, R₁₅, and R₁₆ are each independently hydrogen, or unsubstituted orsubstituted alkyl, aryl, arylalkyl, or cycloalkyl.

[0117] In another embodiment, R₃ is selected from the group consistingof substituted and unsubstituted phenyl, pyridyl, pyrimidyl,pyridazinyl, pyrazinal, pyrrolyl, triazolyl, thioazolyl, oxazolyl,oxadiazolyl, pyrazolyl, furanyl, methylenedioxyphenyl, and thiophenyl.When R₃ is phenyl, it may be substituted with, for example, hydroxyl,alkoxy (e.g., methoxy), alkyl (e.g., tolyl), and halogen, (e.g., o-, m-,or p-fluorophenyl or o-, m-, or p-chlorophenyl). Advantageously, R₃ maybe 2-, 3-, or 4- pyridyl or 2- or 3-pyrimidyl.

[0118] The invention also pertains to a deazapurine wherein R₆ ishydrogen or C₁-C₃ alkyl. Preferably, R₆ is hydrogen.

[0119] The invention also includes deazapurines wherein R₁ is hydrogen,and R₂ is substituted or unsubstituted alkyl or alkoxy, substituted orunsubstituted alkylamine, arylamine, or alkylarylamine, substituted orunsubstituted aminoalkyl, amino aryl, or aminoalkylaryl, substituted orunsubstituted alkylamide, arylamide or alkylarylamide, substituted orunsubstituted alkylsulfonamide, arylsulfonamide or alkylarylsulfonamide,substituted or unsubstituted alkylurea, arylurea or alkylarylurea,substituted or unsubstituted alkylcarbamate, arylcarbamate oralkylarylcarbamate, or substituted or unsubstituted alkylcarboxylicacid, arylcarboxylic acid or alkylarylcarboxylic acid.

[0120] Preferably, R₂ is substituted or unsubstituted cycloalkyl, e.g.,mono- or dihydroxy-substituted cyclohexyl or cyclopentyl (preferably,monohydroxy-substituted cyclohexyl or monohydroxy-substitutedcyclopentyl).

[0121] Advantageously, R₂ may be of the following formula:

[0122] wherein A is C₁-C₆ alkyl, C₃-C₇ cycloalkyl, a chain of one toseven atoms, or a ring of three to seven atoms, optionally substitutedwith C₁-C₆ alkyl, halogens, hydroxyl, carboxyl, thiol, or amino groups;wherein B is methyl, N(Me)₂, N(Et)₂, NHMe, NHEt, (CH₂)_(r)NH₃+,NH(CH₂)_(r)CH₃, (CH₂)_(r)NH₂, (CH₂)_(r)CHCH₃NH₂, (CH₂)_(r)NHMe,(CH₂)_(r)OH, CH₂CN, (CH₂)_(m)CO₂H, CHR₁₈R₁₉, or CHMeOH, wherein r is aninteger from 0 to 2, m is 1 or 2, R₁₈ is alkyl, R₁₉ is NH₃+ or CO₂H orR₁₈ and R₁₉ together are:

[0123] wherein p is 2 or 3; and R₁₇ is C₁-C₆ alkyl, C₃-C₇ cycloalkyl, achain of one to seven atoms, or a ring of three to seven atoms,optionally substituted with C₁-C₆ alkyl, halogens, hydroxyl, carboxyl,thiol, or amino groups.

[0124] Advantageously, A is unsubstituted or substituted C₁-C₆ alkyl. Bmay be unsubstituted or unsubstituted C₁-C₆ alkyl.

[0125] In a preferred embodiment, R₂ is of the formula —A—NHC(═O)B. In aparticularly advantageous embodiment, A is —CH₂CH₂— and B is methyl.

[0126] The compounds of the invention may comprise water-solubleprodrugs which are metabolized in vivo to an active drug, e.g., byesterase catalyzed hydrolysis. Examples of potential prodrugs includedeazapurines with, for example, R₂ as cycloalkyl substituted with—OC(O)(Z)NH₂, wherein Z is a side chain of a naturally or unnaturallyoccurring amino acid, or analog thereof, an α, β, γ, or ω amino acid, ora dipeptide. Preferred amino acid side chains include those of glycine,alanine, valine, leucine, isoleucine, lysine, α-methylalanine,aminocyclopropane carboxylic acid, azetidine-2-carboxylic acid,β-alanine, γ-aminobutyric acid, alanine-alanine, or glycine-alanine.

[0127] In another embodiment, R₁ and R₂ together are:

[0128] wherein n is 1 or 2, and wherein the ring may be optionallysubstituted with one or more hydroxyl, amino, thiol, carboxyl, halogen,CH₂OH, CH₂NHC(═O)alkyl, or CH₂NHC(═O)NHalkyl groups. Preferably, n is 1or 2 and said ring is substituted with —NHC(═O)alkyl.

[0129] In one advantageous embodiment, R₁ is hydrogen, R₂ is substitutedor unsubstituted C₁-C₆ alkyl, R₃ is substituted or unsubstituted phenyl,R₄ is hydrogen, L is hydrogen or substituted or unsubstituted C₁-C₆alkyl, Q is O, S or NR₇, wherein R₇ is hydrogen or substituted orunsubstituted C₁-C₆ alkyl, and W is substituted or unsubstituted aryl.Preferably, R₂ is —A—NHC(═O)B, wherein A and B are each independentlyunsubstituted or substituted C₁-C₄ alkyl. For example, A may be CH₂CH₂.B may be, for example, alkyl (e.g., methyl), or aminoalkyl (e.g.,aminomethyl). Preferably, R₃ is unsubstituted phenyl and L is hydrogen.R₆ may be methyl or preferably, hydrogen. Preferably, Q is O, S, or NR₇wherein R₇ is hydrogen or substituted or unsubstituted C₁-C₆ alkyl,e.g., methyl. W is unsubstituted or substituted phenyl (e.g., alkoxy,halogen substituted). Preferably, W is p-fluorophenyl, p-chlorophenyl,or p-methoxyphenyl. W may also be heteroaryl, e.g., 2-pyridyl.

[0130] In a particularly preferred embodiment, the deazapurine is4-(2-acetylaminoethyl)amino-6-phenoxymethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.

[0131] In a particularly preferred embodiment, the deazapurine is4-(2-acetylaminoethyl)amino-G-(4-fluorophenoxy)methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.

[0132] In a particularly preferred embodiment, the deazapurine is4-(2-acetylaminoethyl)amino-6-(4-chlorophenoxy)methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.

[0133] In a particularly preferred embodiment, the deazapurine is4-(2-acetylaminoethyl)amino-6-(4-methoxyphenoxy)methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.

[0134] In a particularly preferred embodiment, the deazapurine is4-(2-acetylaminoethyl)amino-6-(2-pyridyloxy)methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.

[0135] In a particularly preferred embodiment, the deazapurine is4-(2-acetylaminoethyl)amino-6-(N-phenylamino)methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.

[0136] In a particularly preferred embodiment, the deazapurine is4-(2-acetylaminoethyl)amino-6-(N-methyl-N-phenylamino)methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.

[0137] In a particularly preferred embodiment, the deazapurine is4-(2-N′-methylureaethyl)amino-6-phenoxymethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.

[0138] The invention further pertains to a method for inhibiting theactivity of an adenosine receptor (e.g., an A_(2b) adenosine receptor)in a cell by contacting the cell with a compound of the invention.Preferably, the compound is an antagonist of the receptor.

[0139] The invention also pertains to a method for treating agastrointestinal disorder (e.g., diarrhea) in an animal by administeringto an animal an effective amount of a compound of the invention (e.g.,an antagonist of A_(2b)). Preferably, the animal is a human.

[0140] In another embodiment, the invention relates to a pharmaceuticalcomposition containing an N-6 substituted 7-deazapurine of the inventionand a pharmaceutically acceptable carrier.

[0141] The invention also pertains to a method for treating a N-6substituted 7-deazapurine responsive state in an animal, byadministering to a mammal a therapeutically effective amount of adeazapurine of the invention, such that treatment of a N-6 substituted7-deazapurine responsive state in the animal occurs. Advantageously, thedisease state may be a disorder mediated by adenosine. Examples ofpreferred disease states include: central nervous system disorders,cardiovascular disorders, renal disorders, inflammatory disorders,allergic disorders, gastrointestinal disorders, eye disorders, andrespiratory disorders.

[0142] The term “alkyll” refers to the radical of saturated aliphaticgroups, including straight-chain alkyl groups, branched-chain alkylgroups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkylgroups, and cycloalkyl substituted alkyl groups. The term alkyl furtherincludes alkyl groups, which can further include oxygen, nitrogen,sulfur or phosphorous atoms replacing one or more carbons of thehydrocarbon backbone, e.g., oxygen, nitrogen, sulfur or phosphorousatoms. In preferred embodiments, a straight chain or branched chainalkyl has 30 or fewer carbon atoms in its backbone (e.g., C₁-C₃₀ forstraight chain, C₃-C₃₀ for branched chain), and more preferably 20 orfewer. Likewise, preferred cycloalkyls have from 4-10 carbon atoms intheir ring structure, and more preferably have 5, 6 or 7 carbons in thering structure.

[0143] Moreover, the term alkyl as used throughout the specification andclaims is intended to include both “unsubstituted alkyls” and“substituted alkyls”, the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example,halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,phosphinato, cyano, amino (including alkyl amino, dialkylamino,arylamino, diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Itwill be understood by those skilled in the art that the moietiessubstituted on the hydrocarbon chain can themselves be substituted, ifappropriate. Cycloalkyls can be further substituted, e.g., with thesubstituents described above. An “alkylaryl” moiety is an alkylsubstituted with an aryl (e.g., phenylmethyl (benzyl)). The term “alkyl”also includes unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but that contain atleast one double or triple bond respectively.

[0144] The term “aryl” as used herein, refers to the radical of arylgroups, including 5- and 6-membered single-ring aromatic groups that mayinclude from zero to four heteroatoms, for example, benzene, pyrrole,furan, thiophene, imidazole, benzoxazole, benzothiazole, triazole,tetrazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, andthe like. Aryl groups also include polycyclic fused aromatic groups suchas naphthyl, quinolyl, indolyl, and the like. Those aryl groups havingheteroatoms in the ring structure may also be referred to as “arylheterocycles”, “heteroaryls” or “heteroaromatics”. The aromatic ring canbe substituted at one or more ring positions with such substituents asdescribed above, as for example, halogen, hydroxyl, alkoxy,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato,cyano, amino (including alkyl amino, dialkylamino, arylamino,diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety Arylgroups can also be fused or bridged with alicyclic or heterocyclic ringswhich are not aromatic so as to form a polycycle (e.g., tetralin).

[0145] The terms “alkenyl” and “alkynyl” refer to unsaturated aliphaticgroups analogous in length and possible substitution to the alkylsdescribed above, but that contain at least one double or triple bondrespectively. For example, the invention contemplates cyano andpropargyl groups.

[0146] Unless the number of carbons is otherwise specified, “loweralkyl” as used herein means an alkyl group, as defined above, but havingfrom one to ten carbons, more preferably from one to six carbon atoms inits backbone structure, even more preferably one to three carbon atomsin its backbone structure. Likewise, “lower alkenyl” and “lower alkynyl”have similar chain lengths.

[0147] The terms “alkoxyalkyl”, “polyaminoalkyl” and “thioalkoxyalkyl”refer to alkyl groups, as described above, which further include oxygen,nitrogen or sulfur atoms replacing one or more carbons of thehydrocarbon backbone, e.g., oxygen, nitrogen or sulfur atoms.

[0148] The terms “polycyclyl” or “polycyclic radical” refer to theradical of two or more cyclic rings (e.g., cycloalkyls, cycloalkenyls,cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbonsare common to two adjoining rings, e.g., the rings are “fused rings”.Rings that are joined through non-adjacent atoms are termed “bridged”rings. Each of the rings of the polycycle can be substituted with suchsubstituents as described above, as for example, halogen, hydroxyl,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,phosphinato, cyano, amino (including alkyl amino, dialkylamino,arylamino, diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkyl, alkylaryl, or an aromatic or heteroaromatic moiety.

[0149] The term “heteroatom” as used herein means an atom of any elementother than carbon or hydrogen. Preferred heteroatoms are nitrogen,oxygen, sulfur and phosphorus.

[0150] The term “amino acids” includes naturally and unnaturallyoccurring amino acids found in proteins such as glycine, alanine,valine, cysteine, leucine, isoleucine, serine, threonine, methionine,glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine,proline, histidine, phenylalanine, tyrosine, and tryptophan. Amino acidanalogs include amino acids with lengthened or shortened side chains orvariant side chains with appropriate functional groups. Amino acids alsoinclude D and L stereoisomers of an amino acid when the structure of theamino acid admits of stereoisomeric forms. The term “dipeptide” includestwo or more amino acids linked together. Preferably, dipeptides are twoamino acids linked via a peptide linkage. Particularly preferreddipeptides include, for example, alanine-alanine and glycine-alanine.

[0151] It will be noted that the structure of some of the compounds ofthis invention includes asymmetric carbon atoms and thus occur asracemates and racemic mixtures, single enantiomers, diastereomericmixtures and individual diastereomers. All such isomeric forms of thesecompounds are expressly included in this invention. Each stereogeniccarbon may be of the R or S configuration. It is to be understoodaccordingly that the isomers arising from such asymmetry (e.g., allenantiomers and diastereomers) are included within the scope of thisinvention, unless indicated otherwise. Such isomers can be obtained insubstantially pure form by classical separation techniques and bystereochemically controlled synthesis.

[0152] The invention further pertains to pharmaceutical compositions fortreating a N-6 substituted 7-deazapurine responsive state in a mammal,e.g., respiratory disorders (e.g., asthma, bronchitis, chronicobstructive pulmonary disorder, and allergic rhinitis), renal disorders,gastrointestinal disorders, and eye disorders. The pharmaceuticalcomposition includes a therapeutically effective amount of a N-6substituted 7-deazapurine, described supra, and a pharmaceuticallyacceptable carrier. It is to be understood, that all of the deazapurinesdescribed above are included for therapeutic treatment. It is to befurther understood that the deazapurines of the invention can be usedalone or in combination with other deazapurines of the invention or incombination with additional therapeutic compounds, such as antibiotics,antiinflammatories, or anticancer agents, for example.

[0153] The term “antibiotic” is art recognized and is intended toinclude those substances produced by growing microorganisms andsynthetic derivatives thereof, which eliminate or inhibit growth ofpathogens and are selectively toxic to the pathogen while producingminimal or no deleterious effects upon the infected host subject.Suitable examples of antibiotics include, but are not limited to, theprinciple classes of aminoglycosides, cephalosporins, chloramphenicols,fuscidic acids, macrolides, penicillins, polymixins, tetracyclines andstreptomycins.

[0154] The term “antiinflammatory” is art recognized and is intended toinclude those agents which act on body mechanisms, without directlyantagonizing the causative agent of the inflammation such asglucocorticoids, aspirin, ibuprofen, NSAIDS, etc.

[0155] The term “anticancer agent” is art recognized and is intended toinclude those agents which diminish, eradicate, or prevent growth ofcancer cells without, preferably, adversely affecting otherphysiological functions. Representative examples include cisplatin andcyclophosphamide.

[0156] When the compounds of the present invention are administered aspharmaceuticals, to humans and mammals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

[0157] The phrase “pharmaceutically acceptable carrier” as used hereinmeans a pharmaceutically acceptable material, composition or vehicle,such as a liquid or solid filler, diluent, excipient, solvent orencapsulating material, involved in carrying or transporting acompound(s) of the present invention within or to the subject such thatit can performs its intended function. Typically, such compounds arecarried or transported from one organ, or portion of the body, toanother organ, or portion of the body. Each carrier must be “acceptable”in the sense of being compatible with the other ingredients of theformulation and not injurious to the patient. Some examples of materialswhich can serve as pharmaceutically acceptable carriers include: sugars,such as lactose, glucose and sucrose; starches, such as corn starch andpotato starch; cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatin; talc; excipients, such as cocoa butter andsuppository waxes; oils, such as peanut oil, cottonseed oil, saffloweroil, sesame oil, olive oil, corn oil and soybean oil; glycols, such aspropylene glycol; polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol; phosphate buffer solutions; and other non-toxiccompatible substances employed in pharmaceutical formulations.

[0158] As set out above, certain embodiments of the present compoundscan contain a basic functional group, such as amino or alkylamino, andare, thus, capable of forming pharmaceutically acceptable salts withpharmaceutically acceptable acids. The term “pharmaceutically acceptablesalts” in this respect, refers to the relatively non-toxic, inorganicand organic acid addition salts of compounds of the present invention.These salts can be prepared in situ during the final isolation andpurification of the compounds of the invention, or by separatelyreacting a purified compound of the invention in its free base form witha suitable organic or inorganic acid, and isolating the salt thusformed. Representative salts include the hydrobromide, hydrochloride,sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate,palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts and the like.(See, e.g., Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci.66:1-19).

[0159] A skilled artisan will know that metabolism of the compoundsdisclosed herein in a subject produces certain biologically activemetabolites which can serve as drugs.

[0160] In other cases, the compounds of the present invention maycontain one or more acidic functional groups and, thus, are capable offorming pharmaceutically acceptable salts with pharmaceuticallyacceptable bases. The term “pharmaceutically acceptable salts” in theseinstances refers to the relatively non-toxic, inorganic and organic baseaddition salts of compounds of the present invention. These salts canlikewise be prepared in situ during the final isolation and purificationof the compounds, or by separately reacting the purified compound in itsfree acid form with a suitable base, such as the hydroxide, carbonate orbicarbonate of a pharmaceutically acceptable metal cation, with ammonia,or with a pharmaceutically acceptable organic primary, secondary ortertiary amine. Representative alkali or alkaline earth salts includethe lithium, sodium, potassium, calcium, magnesium, and aluminum saltsand the like. Representative organic amines useful for the formation ofbase addition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like.

[0161] The term “pharmaceutically acceptable esters” refers to therelatively non-toxic, esterified products of the compounds of thepresent invention. These esters can be prepared in situ during the finalisolation and purification of the compounds, or by separately reactingthe purified compound in its free acid form or hydroxyl with a suitableesterifying agent. Carboxylic acids can be converted into esters viatreatment with an alcohol in the presence of a catalyst. Hydroxylcontaining derivatives can be converted into esters via treatment withan esterifying agent such as alkanoyl halides. The term is furtherintended to include lower hydrocarbon groups capable of being solvatedunder physiological conditions, e.g., alkyl esters, methyl, ethyl andpropyl esters. (See, for example, Berge et al., supra.)

[0162] The invention further contemplates the use of prodrugs which areconverted in vivo to the therapeutic compounds of the invention (see,e.g., R. B. Silverman, 1992, “The Organic Chemistry of Drug Design andDrug Action”, Academic Press, Chapter 8). Such prodrugs can be used toalter the biodistribution (e.g., to allow compounds which would nottypically enter the reactive site of the protease) or thepharmacokinetics of the therapeutic compound. For example, a carboxylicacid group, can be esterified, e.g., with a methyl group or an ethylgroup to yield an ester. When the ester is administered to a subject,the ester is cleaved, enzymatically or non-enzymatically, reductively orhydrolytically, to reveal the anionic group. An anionic group can beesterified with moieties (e.g., acyloxymethyl esters) which are cleavedto reveal an intermediate compound which subsequently decomposes toyield the active compound. In another embodiment, the prodrug is areduced form of a sulfate or sulfonate, e.g., a thiol, which is oxidizedin vivo to the therapeutic compound. Furthermore, an anionic moiety canbe esterified to a group which is actively transported in vivo, or whichis selectively taken up by target organs. The ester can be selected toallow specific targeting of the therapeutic moieties to particularreactive sites, as described below for carrier moieties.

[0163] Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

[0164] Examples of pharmaceutically acceptable antioxidants include:water soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and metalchelating agents, such as citric acid, ethylenediamine tetraacetic acid(EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0165] Formulations of the present invention include those suitable fororal, nasal, topical, transdermal, buccal, sublingual, rectal, vaginaland/or parenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willgenerally be that amount of the compound which produces a therapeuticeffect. Generally, out of one hundred per cent, this amount will rangefrom about 1 per cent to about ninety-nine percent of active ingredient,preferably from about 5 per cent to about 70 per cent, most preferablyfrom about 10 per cent to about 30 per cent.

[0166] Methods of preparing these formulations or compositions includethe step of bringing into association a compound of the presentinvention with the carrier and, optionally, one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association a compound of the present inventionwith liquid carriers, or finely divided solid carriers, or both, andthen, if necessary, shaping the product.

[0167] Formulations of the invention suitable for oral administrationmay be in the form of capsules, cachets, pills, tablets, lozenges (usinga flavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

[0168] In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: fillers or extenders, such as starches, lactose, sucrose,glucose, mannitol, and/or silicic acid; binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; humectants, such as glycerol; disintegratingagents, such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate; solutionretarding agents, such as paraffin; absorption accelerators, such asquaternary ammonium compounds; wetting agents, such as, for example,cetyl alcohol and glycerol monostearate; absorbents, such as kaolin andbentonite clay; lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and coloring agents. In the case of capsules, tabletsand pills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard-filled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

[0169] A tablet may be made by compression or molding, optionally withone or more accessory ingredients. Compressed tablets may be preparedusing binder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

[0170] The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

[0171] Liquid dosage forms for oral administration of the compounds ofthe invention include pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the active ingredient, the liquid dosage forms may contain inertdilutents commonly used in the art, such as, for example, water or othersolvents, solubilizing agents and emulsifiers, such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (inparticular, cottonseed, groundnut, corn, germ, olive, castor and sesameoils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof.

[0172] Besides inert dilutents, the oral compositions can also includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, coloring, perfuming and preservative agents.

[0173] Suspensions, in addition to the active compounds, may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

[0174] Formulations of the pharmaceutical compositions of the inventionfor rectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

[0175] Formulations of the present invention which are suitable forvaginal administration also include pessaries, tampons, creams, gels,pastes, foams or spray formulations containing such carriers as areknown in the art to be appropriate.

[0176] Dosage forms for the topical or transdermal administration of acompound of this invention include powders, sprays, ointments, pastes,creams, lotions, gels, solutions, patches and inhalants. The activecompound may be mixed under sterile conditions with a pharmaceuticallyacceptable carrier, and with any preservatives, buffers, or propellantswhich may be required.

[0177] The ointments, pastes, creams and gels may contain, in additionto an active compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

[0178] Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

[0179] Transdermal patches have the added advantage of providingcontrolled delivery of a compound of the present invention to the body.Such dosage forms can be made by dissolving or dispersing the compoundin the proper medium. Absorption enhancers can also be used to increasethe flux of the compound across the skin. The rate of such flux can becontrolled by either providing a rate controlling membrane or dispersingthe active compound in a polymer matrix or gel.

[0180] Ophthalmic formulations, eye ointments, powders, solutions andthe like, are also contemplated as being within the scope of thisinvention. Preferably, the pharmaceutical preparation is an ophthalmicformulation (e.g., an periocular, retrobulbar or intraocular injectionformulation, a systemic formulation, or a surgical irrigating solution).

[0181] The ophthalmic formulations of the present invention may includeone or more deazapurines and a pharmaceutically acceptable vehicle.Various types of vehicles may be used. The vehicles will generally beaqueous in nature. Aqueous solutions are generally preferred, based oncase of formulation, as well as a patient's ability to easily administersuch compositions by means of instilling one to two drops of thesolutions in the affected eyes. However, the deazapurines of the presentinvention may also be readily incorporated into other types ofcompositions, such as suspensions, viscous or semi-viscous gels or othertypes of solid or semi-solid compositions. The ophthalmic compositionsof the present invention may also include various other ingredients,such as buffers, preservatives, co-solvents and viscosity buildingagents.

[0182] An appropriate buffer system (e.g., sodium phosphate, sodiumacetate or sodium borate) may be added to prevent pH drift under storageconditions.

[0183] Ophthalmic products are typically packaged in multidose form.Preservatives are thus required to prevent microbial contaminationduring use. Suitable preservatives include: benzalkonium chloride,thimerosal, chlorobutanol, methyl paraben, propyl paraben, phenylethylalcohol, edetate disodium, sorbic acid, polyquaternium-l, or otheragents known to those skilled in the art. Such preservatives aretypically employed at a level of from 0.001 to 1.0% weight/volume (“%w/v”).

[0184] When the deazapurines of the present invention are administeredduring intraocular surgical procedures, such as through retrobulbar orperiocular injection and intraocular perfusion or injection, the use ofbalanced salt irrigating solutions as vehicles are most preferred. BSS®Sterile Irrigating Solution and BSS Plus® Sterile Intraocular IrrigatingSolution (Alcon Laboratories, Inc., Fort Worth, Tex., USA) are examplesof physiologically balanced intraocular irrigating solutions. The lattertype of solution is described in U.S. Pat. No. 4,550,022 (Garabedian, etal.), the entire contents of which are hereby incorporated in thepresent specification by reference. Retrobulbar and periocularinjections are known to those skilled in the art and are described innumerous publications including, for example, Ophthalmic Surgery:Principles of Practice, Ed., G. L. Spaeth. W. B. Sanders Co.,Philadelphia, Pa., U.S.A., pages 85-87 (1990).

[0185] As indicated above, use of deazapurines to prevent or reducedamage to retinal and optic nerve head tissues at the cellular level isa particularly important aspect of one embodiment of the invention.Ophthalmic conditions which may be treated include, but are not limitedto, retinopathies, macular degeneration, ocular ischemia, glaucoma, anddamage associated with injuries to ophthalmic tissues, such as ischemiareperfusion injuries, photochemical injuries, and injuries associatedwith ocular surgery, particularly injuries to the retina or optic nervehead by exposure to light or surgical instruments. The compounds mayalso be used as an adjunct to ophthalmic surgery, such as by vitreal orsubconjunctival injection following ophthalmic surgery. The compoundsmay be used for acute treatment of temporary conditions, or may beadministered chronically, especially in the case of degenerativedisease. The compounds may also be used prophylactically, especiallyprior to ocular surgery or noninvasive ophthalmic procedures, or othertypes of surgery.

[0186] Pharmaceutical compositions of this invention suitable forparenteral administration comprise one or more compounds of theinvention in combination with one or more pharmaceutically acceptablesterile isotonic aqueous or nonaqueous solutions, dispersions,suspensions or emulsions, or sterile powders which may be reconstitutedinto sterile injectable solutions or dispersions just prior to use,which may contain antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents.

[0187] Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

[0188] These compositions may also contain adjuvants such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

[0189] In some cases, in order to prolong the effect of a drug, it isdesirable to slow the absorption of the drug from subcutaneous orintramuscular injection. This may be accomplished by the use of a liquidsuspension of crystalline or amorphous material having poor watersolubility. The rate of absorption of the drug then depends upon itsrate of dissolution which, in turn, may depend upon crystal size andcrystalline form. Alternatively, delayed absorption of aparenterally-administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle.

[0190] Injectable depot forms are made by forming microencapsulematrices of the subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

[0191] The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given by formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Oral administration is preferred.

[0192] The phrases “parenteral administration” and “administeredparenterally” as used herein means modes of administration other thanenteral and topical administration, usually by injection, and includes,without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion.

[0193] The phrases “systemic administration,” “administeredsystematically,” “peripheral administration” and “administeredperipherally” as used herein mean the administration of a compound, drugor other material other than directly into the central nervous system,such that it enters the patient's system and, thus, is subject tometabolism and other like processes, for example, subcutaneousadministration.

[0194] These compounds may be administered to humans and other animalsfor therapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

[0195] Regardless of the route of administration selected, the compoundsof the present invention, which may be used in a suitable hydrated form,and/or the pharmaceutical compositions of the present invention, areformulated into pharmaceutically acceptable dosage forms by conventionalmethods known to those of skill in the art.

[0196] Actual dosage levels of the active ingredients in thepharmaceutical compositions of this invention may be varied so as toobtain an amount of the active ingredient which is effective to achievethe desired therapeutic response for a particular patient, composition,and mode of administration, without being toxic to the patient.

[0197] The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compound employed, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well known in the medical arts.

[0198] A physician or veterinarian having ordinary skill in the art canreadily determine and prescribe the effective amount of thepharmaceutical composition required For example, the physician orveterinarian could start doses of the compounds of the inventionemployed in the pharmaceutical composition at levels lower than thatrequired in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved.

[0199] In general, a suitable daily dose of a compound of the inventionwill be that amount of the compound which is the lowest dose effectiveto produce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, intravenous andsubcutaneous doses of the compounds of this invention for a patient,when used for the indicated analgesic effects, will range from about0.0001 to about 200 mg per kilogram of body weight per day, morepreferably from about 0.01 to about 150 mg per kg per day, and stillmore preferably from about 0.2 to about 140 mg per kg per day.

[0200] If desired, the effective daily dose of the active compound maybe administered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

[0201] While it is possible for a compound of the present invention tobe administered alone, it is preferable to administer the compound as apharmaceutical composition.

[0202] The present invention also pertains to packaged pharmaceuticalcompositions for treating a N-6 substituted 7 deazapurine responsivestate, e.g., undesirable increased adenosine receptor activity in amammal. The packaged pharmaceutical compositions include a containerholding a therapeutically effective amount of at least one deazapurineas described supra and instructions for using the deazapurine fortreating the deazapurine responsive state in the mammal.

[0203] The deazapurines of the invention can be prepared using standardmethods for organic synthesis. Deazapurines can be purified by reversephase HPLC, chromatography, recrystallization, etc. and their structuresconfirmed by mass spectral analysis, elemental analysis, IR and/or NMRspectroscopy.

[0204] Typically, synthesis of the intermediates as well as thedeazapurines of the invention is performed in solution. The addition andremoval of one or more protecting group is also typical practice and isknown to those skilled in the art. Typical synthetic schemes for thepreparation of deazapurine intermediates of the invention are outlinedbelow in Scheme I.

[0205] This invention also provides a compound having the structure:

[0206] wherein NR₁R₂ is a substituted or unsubstituted 4-8 memberedring;

[0207] wherein Ar is a substituted or unsubstituted four to six memberedring;

[0208] wherein R₄ is H, alkyl, substituted alkyl, aryl, arylalkyl,amino, substituted aryl, wherein said substituted alkyl is —C(R₈)(R₉)XR₆, wherein X is O, S, or NR₇, wherein R₈ and R₉ are eachindependently H or alkyl, wherein R₆ and R₇ are each independently alkylor cycloalkyl, or R₆, R₇ and the nitrogen together form a substituted orunsubstituted ring of between 4 and 7 members.

[0209] wherein R₅ is H, alkyl, substituted alkyl, or cycloalkyl;

[0210] with the proviso that NR₁R₂ is not 3-acetamido piperadino,3-hydroxy pyrrolidino, 3-methyloxy carbonylmethyl pyrrolidino,3-aminocarbonylmethyl, or pyrrolidino; with the proviso that NR₁R₂ is3-hydroxymethyl piperadino only when Ar is 4-pyridyl.

[0211] In one embodiment of the compound, Ar is a substituted orunsubstituted four to six membered ring, phenyl, pyrrole, thiophene,furan, thiazole, imidazole, pyrazole, 1,2,4-triazole, pyridine,2(1H)-pyridone, 4(1H)-pyridone, pyrazine, pyrimidine, pyridazine,isothiazole, isoxazole, oxazole, tetrazole, naphthalene, tetralin,naphthyridine, benzofuran, benzothiophene, indole, 2,3-dihydroindole,1H-indole, indoline, benzopyrazole, 1,3-benzodioxole, benzoxazole,purine, coumarin, chromone, quinoline, tetrahydroquinoline,isoquinoline, benzimidazole, quinazoline, pyrido[2,3-b]pyrazine,pyrido[3,4-b]pyrazine, pyrido[3,2-c]pyridazine, purido[3,4-b]-pyridine,1H-pyrazole[3,4-d]pyrimidine, pteridine, 2(1H)-quinolone,1(2H)-isoquinolone, 1,4-benzisoxazine, benzothiazole, quinoxaline,quinoline-N-oxide, isoquinoline-N-oxide, quinoxaline-N-oxide,quinazoline-N-oxide, benzoxazine, phthalazine, cinnoline, or having astructure:

[0212] wherein Y is carbon or nitrogen;

[0213] wherein R₃ is H, substituted or unsubstituted alkyl, substitutedor unsubstituted aryl, halogen, methoxy, methyl amino, methyl thio;

[0214] In another embodiment of the compound, the compound has thestructure:

[0215] wherein m is 1 or 2; wherein R_(A) and R_(B) are eachindependently be H, —OH, —CH₂OH, —CH₂CH₂OH, —C(═O)NH₂, a heteroatom, or—C(═O)NR₃R₃′; wherein R₃ is aryl, substituted aryl, or heteroaryl;wherein R₃′ is alkyl, or XR₃″, wherein X is O, or N and R″ issubstituted alkyl or aryl.

[0216] In another embodiment of the compound, R₁R₂N is(D)-2-aminocarbonyl pyrrolidino, (D)-2-hydroxymethyl pyrrolidino,(D)-2-hydroxymethyl-trans-4-hydroxy pyrrolidino, piperazino, or3-hydroxymethyl piperadino.

[0217] In another embodiment of the compound, the compound has thestructure:

[0218] wherein m is 0, 1, 2, or 3; wherein Y is O, S, or NR, wherein Ris R_(A) or R_(B); wherein R_(A) and R_(B) are each independently be H,—OH, —CH₂OH, —CH₂CH₂OH, —C(═O)NH₂, a heteroatom, or —C(═O)NR₃R₃′;wherein R₃ is aryl, substituted aryl, or heteroaryl; wherein R₃′ isalkyl, or XR₃″, wherein X is O, or N and R″ is substituted alkyl oraryl.

[0219] In another embodiment of the compound, the compound has thestructure:

[0220] In another embodiment of the compound, the compound has thestructure:

[0221] In another embodiment of the compound, the compound has thestructure:

[0222] In another embodiment of the compound, the compound has thestructure:

[0223] In another embodiment of the compound, the compound has thestructure:

[0224] In another embodiment of the compound, the compound has thestructure:

[0225] In another embodiment of the compound, the compound has thestructure:

[0226] In another embodiment of the compound, the compound has thestructure:

[0227] In yet another embodiment of the compound, the compound has thestructure:

[0228] In a further embodiment of the compound, the compound has thestructure:

[0229] This invention further provides a compound having the structure(V)

[0230] wherein R is H, or methyl.

[0231] In one embodiment of the compound V, the compound has thestructure:

[0232] In another embodiment of the compound V, the compound has thestructure:

[0233] This invention also provides a method for treating a diseaseassociated with A_(2a) adenosine receptor in a subject, comprisingadministering to the subject a therapeutically effective amount ofcompounds IV, or V.

[0234] In one embodiment of the method, the compound treats saiddiseases by stimulating adenylate cyclase.

[0235] In another embodiment of the method, the subject is a mammal.

[0236] In another embodiment of the method, the mammal is a human.

[0237] In another embodiment of the method, said A_(2a) adenosinereceptor is associated with Parkinson's disease and diseases associatedwith locomotor activity, vasodilation, platelet inhibition, neutrophilsuperoxide generation, cognitive disorder, or senile dementia.

[0238] Diseases associated with adenosine A₁, A_(2a), A_(2b) and A₃receptors are disclosed in WO 99/06053 and WO-09822465, WO-09705138,WO-09511681, WO-09733879, JP-09291089, PCT/US98/16053 and U.S. Pat. No.5,516,894, the entire content of which are fully incorporate herein byreference.

[0239] This invention also provides a water-soluble prodrug of compoundsIV, or V; wherein said water-soluble prodrug that is metabolized in vivoto produce an active drug which selectively inhibit A_(2a) adenosinereceptor.

[0240] In one embodiment of the prodrug, said prodrug is metabolized invivo by esterase catalyzed hydrolysis.

[0241] This invention also provides a pharmaceutical compositioncomprising the prodrug and a pharmaceutically acceptable carrier.

[0242] This invention also provides a method for inhibiting the activityof an A_(2a) adenosine receptor in a cell, which comprises contactingsaid cell with compounds IV, or V.

[0243] In one embodiment of the method, the compound is an antagonist ofsaid A_(2a) adenosine receptor.

[0244] In another embodiment of the pharmaceutical composition, saidpharmaceutical composition is an ophthalmic formulation.

[0245] In another embodiment of the pharmaceutical composition, saidpharmaceutical composition is an periocular, retrobulbar or intraocularinjection formulation.

[0246] In another embodiment of the pharmaceutical composition, saidpharmaceutical composition is a systemic formulation.

[0247] This invention also provides a method for treating agastrointestinal disorder in an subject, comprising administering to thean effective amount of compounds IV, or V.

[0248] In one embodiment of the method, said disorder is diarrhea.

[0249] In another embodiment of the method, the subject is a human.

[0250] In another embodiment of the method, the compound is anantagonist of A_(2a) adenosine receptors.

[0251] This invention further provides a method for treating respiratorydisorder in a subject, comprising administering to the subject aneffective amount of compounds IV, or V.

[0252] In one embodiment of the method, said disorder is asthma, chronicobstructive pulmonary disease, allergic rhinitis, or an upperrespiratory disorder.

[0253] In another embodiment of the method, the subject is a human.

[0254] In another embodiment of the method, said compound is anantagonist of A_(2a) adenosine receptors.

[0255] This invention also provides a method for treating damage to theeye of a subject which comprises administering to said subject aneffective amount of compounds IV, or V.

[0256] In one embodiment of the method, said damage comprises retinal oroptic nerve head damage.

[0257] In another embodiment of the method, said damage is acute orchronic.

[0258] In another embodiment of the method, said damage is the result ofglaucoma, edema, ischemia, hypoxia or trauma.

[0259] In another embodiment of the method, the subject is a human.

[0260] In another embodiment of the method, the compound is anantagonist of A_(2a) adenosine receptors.

[0261] This invention also provide a pharmaceutical compositioncomprising a therapeutically effective amount of compounds IV, or V anda pharmaceutically acceptable carrier.

[0262] In one embodiment of the pharmaceutical composition, saidtherapeutically effective amount is effective to treat Parkinson'sdisease and diseases associated with locomotor activity, vasodilation,platelet inhibition, neutrophil superoxide generation, cognitivedisorder, or senile dementia.

[0263] In another embodiment of the pharmaceutical composition, saidpharmaceutical composition is an ophthalmic formulation.

[0264] In another embodiment of the pharmaceutical composition, saidpharmaceutical composition is an periocular, retrobulbar or intraocularinjection formulation.

[0265] In another embodiment of the pharmaceutical composition, saidpharmaceutical composition is a systemic formulation.

[0266] In another embodiment of the pharmaceutical composition, saidpharmaceutical composition is a surgical irrigating solution.

[0267] This invention also provides a combination therapy forParkinson's disease comprising compounds IV and V, and any of thedopamine enhancers.

[0268] This invention further provides a combinational therapy forcancer comprising compounds IV and V, and any of the cytotoxic agents.

[0269] This invention further provides a combinational therapy forglaucoma, comprising compounds IV or V, and a prostaglandin agonist, amuscrinic agonist, or a β-2 antagonist.

[0270] This invention also provides a packaged pharmaceuticalcomposition for treating a disease associated with A_(2a) adenosinereceptor in a subject, comprising: (a) a container holding atherapeutically effective amount of compounds IV, or V; and (b)instructions for using said compound for treating said disease in asubject.

[0271] This invention also provide a method of preparing compound IV,comprising the steps of

[0272] a)

[0273] wherein P is a removable protecting group;

[0274] b) treating the product of step a) under cyclization conditionsto provide

[0275] c) treating the product of step b) under suitable conditions toprovide R₄; and

[0276] d) treating the chlorinated product of step c) with NHR₁R₂ toprovide

[0277] wherein NR₁R₂ is a substituted or unsubstituted 4-8 memberedring;

[0278] wherein Ar is a substituted or unsubstituted four to six memberedring;

[0279] wherein R₄ is H, alkyl, substituted alkyl, aryl, arylalkyl,amino, substituted aryl, wherein said substituted alkyl is —C(R₈)(R₉)XR₆, wherein X is O, S, or NR₇, wherein R₈ and R₉ are eachindependently H or alkyl, wherein R₆ and R₇ are each independently alkylor cycloalkyl, or R₆, R₇ and the nitrogen together form a substituted orunsubstituted ring of between 4 and 7 members.

[0280] wherein R₅ is H, alkyl, substituted alkyl, or cycloalkyl;

[0281] with the proviso that NR₁R₂ is not 3-acetamido piperadino,3-hydroxy pyrrolidino, 3-methyloxy carbonylmethyl pyrrolidino,3-aminocarbonylmethyl, or pyrrolidino; with the proviso that NR₁R₂ is3-hydroxymethyl piperadino only when Ar is 4-pyridyl.

[0282] This invention further provides a method of preparing compound V,comprising the steps of

[0283] a)

[0284] wherein P is a removable protecting group;

[0285] b) treating the product of step a) under cyclization conditionsto provide

[0286] c) treating the product of step b) under suitable conditions toprovide

[0287] d) treating the chlorinated product of step c) first withdimethylamine and formaldehyde, then with N-methyl benzylamine andfinally with NH₂R₁ to provide

[0288] wherein R₁ is acetomido ethyl; wherein Ar is 4-pyridyl; wherein Ris H, or methyl; wherein R₅ is N-methyl-N-benzyl aminomethyl.

[0289] As used herein, “A compound is A_(2a) selective.” means that acompound has a binding constant to adenosine A_(2a) receptor of at leastfive time higher then that to adenosine A₁, A_(2b), or A₃.

[0290] The invention is further illustrated by the following exampleswhich in no way should be construed as being further limiting. Thecontents of all references, pending patent applications and publishedpatent applications, cited throughout this application, including thosereferenced in the background section, are hereby incorporated byreference. It should be understood that the models used throughout theexamples are accepted models and that the demonstration of efficacy inthese models is predictive of efficacy in humans.

[0291] This invention will be better understood from the ExperimentalDetails which follow. However, one skilled in the art will readilyappreciate that the specific methods and results discussed are merelyillustrative of the invention as described more fully in the claimswhich follow thereafter.

[0292] Experimental Details

[0293] The deazapurines of the invention can be prepared using standardmethods for organic synthesis. Deazapurines can be purified by reversephase HPLC, chromatography, recrystallization, etc. and their structuresconfirmed by mass spectral analysis, elemental analysis, IR and/or NMRspectroscopy.

[0294] Typically, synthesis of the intermediates as well as thedeazapurines of the invention is performed in solution. The addition andremoval of one or more protecting group is also typical practice and isknown to those skilled in the art. Typical synthetic schemes for thepreparation of deazapurine intermediates of the invention are outlinedbelow in Scheme I.

[0295] wherein R₃, R₅ and R₆ are as defined above.

[0296] In general, a protected 2-amino-3-cyano-pyrrole can be treatedwith an acyl halide to form a carboxyamido-3-cyano-pyrrole which can betreated with acidic methanol to effect ring closure to apyrrolo[2,3d]pyrimidine-4(3H)-one (Muller, C. E. et al. J. Med. Chem.40:4396 (1997)). Removal of the pyrrolo protecting group followed bytreatment with a chlorinating reagent, e.g., phosphorous oxychloride,produced substituted or unsubstituted4-chloro-7H-pyrrolo[2,3d]pyrimidines. Treatment of the chloropyrimidinewith amines afforded 7-deazapurines.

[0297] For example, as shown in Scheme I, aN-(1-dl-phenylethyl)-2-amino-3-cyano-pyrrole was treated with an acylhalide in pyridine and dichloromethane. The resultantN-(1-dl-phenylethyl)-2-phenylcarboxyamido-3-cyano-pyrrole was treatedwith a 10:1 mixture of methanol/sulfuric acid to effect ring closure,resulting in a dl-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyrimidine-4(3H)-one.Removal of the phenylethyl group by treatment of the pyrimidine withpolyphosphoric acid (PPA) followed by POCl₃ afforded a key intermediate,the 4-chloro-7H-pyrrolo[2,3d]pyrimidine. Further treatment of the4-chloro-7H-pyrrolo[2,3d]pyrimidine with various amines listed in Table1 gives compounds of formula (I) and (II). TABLE 1 R M⁺ + H R M⁺ + H

343.2 

351.27

343.18

430.35

337.21

359.44

364.19

404.32

330.18

330.45

347.22

339.47

350.28

353.41

344.19

324.45

394.16

359.38

371.12

379.40

359.39

387.41

403.33

344.48

351.49

337.53

330.37

295.2

407.23

321.2

355.45

337.53

441.33

350.2

413.24

343.2

372.48

373.2

307.2

[0298] A general approach to prepare 6-substituted pyrroles is depictedin the following scheme (Scheme II).

[0299] wherein R₁ through R₅ are as defined above.

[0300] Transesterification and alkylation of ethyl cyanoacetate with anα-haloketone affords a ketomethylester. Protection of the ketonefollowed by treatment with an amidine (e.g., alkyl, aryl or alkylaryl)hydrochloride produced the resultant ketal protected pyrimidine. Removalof the protecting group, followed by cyclization and treatment withphosphorous oxychloride afforded the chloride intermediate which couldbe further treated with an amine to afford an amine 6-substitutedpyrrole. Additionally, alkylation of the pyrrole nitrogen can beachieved under art recognized conditions.

[0301] A general approach to prepare 5-substituted pyrroles is depictedin the following scheme (Scheme III).

[0302] wherein R₁ through R₆ are defined as above and R is a removableprotecting group.

[0303] Condensation of malononitrile and an excess of a ketone followedby bromination of the product afforded a mixture of starting material,monobrominated and dibrominated products which were treated with analkylamine, arylamine or alkylarylamine. The resultant amine product wasacylated with an acid chloride and the monacylated pyrrole was cyclizedin the presence of acid to afford the corresponding pyrimidine. Thepyrrole protecting group was removed with polyphosphoric acid andtreated with phosphorous oxychloride to produce a chlorinated product.The chlorinated pyrrole could subsequently be treated with an amine toproduce an amino 5-substituted pyrrole. Alkylation of the pyrrolenitrogen can be achieved under art recognized conditions.

[0304] Schemes IV and V depict methods for preparing the deazapurines 1and 2 of the invention.

[0305] wherein R₅ and R₆ are as described above, e. g., CH₃.

[0306] Specific Preparation of 6-methyl Dyrrolopyrimidines:

[0307] The key reaction toward 6-methylpyrrolopyrimidines (1) [R₅═CH₃]was cyclization of a cyanoacetate with benzamidine to a pyrimidine. Itwas believed methyl cyanoacetate would cyclize more efficiently withbenzamidine to a pyrimidine than the corresponding ethyl ester.Therefore, transesterification and alkylation of ethyl cyanoacetate inthe presence of NaCMe and an excess of an a-haloacetyl moiety, e.g.,chloroacetone, gave the desired methyl ester (3) in 79% yield (SchemeIV). The ketoester (3) was protected as the acetal (4) in 81W yield. Anew cyclization method to the pyrimidine (5) was achieved with anamidine hydrochloride, e.g., benzamidine hydrochloride, with 2equivalents of DBU to afford the 5 in 54% isolated yield. This methodimproves the yield from 20% using the published conditions, whichutilizes NaOMe during the cyclization with guanidine. Cyclization to thepyrrole-pyrimidine (6) was achieved via deprotection of the acetal inaqueous HCl in 78% yield. Reaction of (6) with phosphorous oxychlorideat reflux gave the corresponding 4-chloro derivative (7). Coupling withtrans-4-aminocyclohexanol in dimethyl sulfoxide at 135° C. gave (1) in57% from (7). One skilled in the art will appreciate that choice ofreagents allows for great flexibility in choosing the desiredsubstituent R₅.

[0308] Specific Preparation of 5-methylpyrrolopyrimidines

[0309] Knoevengel condensation of malononitrile and an excess ketone,e.g., acetone in refluxing benzene gave 8 in 50S yield afterdistillation. Bromination of 8 with N-bromosuccinimde in the presence ofbenzoyl peroxide in chloroform yielded a mixture of starting material,mono-(9), and di-brominated products (5/90/5) after distillation (70%).The mixture was reacted with an α-methylalkylamine or α-methylarylamine,e.g., a-methylbenzylamine, to deliver the aminopyrrole (10). Afterpassing through a short silica gelcolumn, the partially purified amine(31% yield) was acylated with an acid chloride, e.g., benzoyl chlorideto deliver mono-(11), and diacylated (12) pyrroles, which were separatedby flash chromatography. Acid hydrolysis of the disubstituted pyrrole(12) generated a combined yield of 29% for the acylpyrrole (11).Cyclization in the presence of concentrated sulphuric acid and DMFyielded (13) (23%), which was deprotected with polyphosphoric acid to(14). Reaction of (14) with phosphorous oxychloride at reflux gave thecorresponding 4-chloro derivative (15). Coupling withtrans-4-aminocyclohexanol in dimethyl sulfoxide at 135° C. gave (2)[R₆═CH₃] in 30% from (14) (See Scheme V). One skilled in the art willappreciate that choice of reagents allows for great flexibility inchoosing the desired substituent R₆.

[0310] Alternative Synthetic Route to R₆-Substituted Pyrroles, e.g.,5-methyl pyrrolopyrimidines:

[0311] This alternative route to R₆-substituted pyrroles, e.g.,5-methylpyrrolopyrimidines, involves transesterification and alkylationof ethyl cyanoacetate to (16) (Scheme VI). The condensation of (16) withbenzamidine hydrochloride with 2 equivalents of DBU affords thepyrimidine (17). Cyclization to the pyrrole-pyrimidine (14) will beachieved via deprotection of the acetal in aqueous HCl. Reaction of (14)with phosphorous oxychloride at reflux gave the corresponding 4-chloroderivative (15). Coupling with trans-4-aminocyclohexanol in dimethylsulfoxide at 135° C. gives 2. This procedure reduces the number ofsynthetic reactions to the target compound (2) from 9 to 4 steps.Moreover, the yield is dramatically improved. Again, one skilled in theart will appreciate that choice of reagents allows for great flexibilityin choosing the desired substituent R₆.

[0312] A general approach to prepare des-methyl pyrrole is depicted inthe following scheme (Scheme VII)

[0313] wherein R₁ through R₃ are defined as above.

[0314] Alkylation of an alkyl cyanoacetate with a diethyl acetal in thepresence of a base afforded a cyano diethyl acetal which was treatedwith an amidine salt to produce a methyl pyrrolopyrimidine precursor.The precursor was chlorinated and treated with an amine to form thedes-methyl pyrrolopyrimidine target as shown above.

[0315] For example, Scheme VIII depicts the synthesis of compound

[0316] Commercially available methyl cyanoacetate was alkylated withbromoacetaldehyde diethyl acetal in the presence of potassium carbonateand NaI to yield (19). Cyclization to the pyrimidine (20) was achievedin two steps. Initially, the pyrimidine-acetal was formed via reactionof (19) with benzamidine hydrochloride with 2 equivalents of DBU. Theresultant pyrimidine-acetal was deprotected without purification withaqueous 1 N HCl and the resultant aldehyde cyclized to thepyrrolo-pyrimidine (20), which was isolated by filtration. Reaction of(20) with phosphorous oxychloride at reflux afforded the corresponding4-chloro derivative (21). Coupling of the chloro derivative withtrans-4-aminocyclohexanol in DMSO at 135° C. gave compound (18) fromcompound (21).

[0317] Schemes II-VIII demonstrate that it is possible to functionalizethe 5- and 6-position of the pyrrolopyrimidine ring. Through the use ofdifferent starting reagents and slight modifications of the abovereaction schemes, various functional groups can be introduced at the 5-and 6-positions in formula (I) and (II). Table 2 illustrates someexamples. TABLE 2 Selected list of 5- and 6-substitutedpyrrolopyrimidines. Starting Reagent R₅ R₆

H

H Substituted Ar

H CH₂C(O)OCH₃

C(O)OCH₃ CH₃

C(O)NHCH₃ CH₃

[0318] The invention is further illustrated by the following exampleswhich in no way should be construed as being further limiting. Thecontents of all references, pending patent applications and publishedpatent applications, cited throughout this application, including thosereferenced in the background section, are hereby incorporated byreference. It should be understood that the models used throughout theexamples are accepted models and that the demonstration of efficacy inthese models is predictive of efficacy in humans.

[0319] Exemplification

[0320] Preparation 1:

[0321] A modification of the alkylation method of Seela and Lüpke wasused.¹ To an ice-cooled (0° C.) solution of ethyl cyanoacetate (6.58 g,58.1 mmol) in MeOH (20 mL) was slowly added a solution of NaOMe (25%w/v; 58.1 mmol). After 10 min, chloroacetone (5 mL; 62.8 mmol) wasslowly added. After 4 hrs, the solvent was removed. The brown oil wasdiluted the EtOAc (100 mL) and washed with H₂O (100 mL). The organicfraction was dried, filtered, and concentrated to a brown oil (7.79 g;79%). The oil (3) (Scheme IV) was a mixture of methyl/ethyl esterproducts (9/1), and was used without further purification. ¹H NMR (200MHz, CDCl₃) δ 4.24 (q, J=7.2 Hz, OCH₂), 3.91 (dd, 1H, J=7.2, 7.0 Hz,CH), 3.62 (s, 3H, OCH₃), 3.42 (dd, 1H, J=15.0, 7.1 Hz, 1×CH₂); 3.02 (dd,1H, J=15.0, 7.0 Hz, 1×CH₂); 2.44 (s, 3H, CH₃), 1.26 (t, J=7.1 Hz,ester-CH₃).

[0322] Preparation 2:

[0323] The procedure of Seela and Lupke was used.¹ Thus, protection ofthe ketone (3) (Scheme IV; 5.0 g, 32.2 mmol) with ethylene glycol (4 mL,64.4 mmol) in the presence of TsOH (100 mg) afforded (4) as an oil(Scheme IV; 5.2 g, 81.0) after flash chromatography (SiO₂; 3/7EtOAc/Hex, R_(f) 0.35). Still contains ^(˜)5% ethyl ester: ¹H NMR (200MHz, CDCl₃) δ_(—)4.24 (q, J=7.2 Hz, OCH₂), 3.98 (s, 4H, 2×acetal-CH₂),3.79 (s, 3H, OCH₃), 3.62 (dd, 1H, J=7.2, 7.0 Hz, CH), 2.48 (dd, 1H,J=15.0, 7.1 Hz, 1×CH₂), 2.32 (dd, 1H, J=15.0, 7.0 Hz, 1×CH₂); 1.35 (s,3H, CH₃), 1.26 (t, J=7.1 Hz, ester-CH₃); MS (ES): 200.1 (M⁺+1).

[0324] Preparation 3:

[0325] A solution of acetal (4) (Scheme IV, 1 g, 5.02 mmol), benzamidine(786 mg, 5.02 mmol), and DBU (1.5 mL, 10.04 mmol) in dry DMF (15 mL) washeated to 85° C. for 15 h. The mixture was diluted with CHCl₃ (30 mL)and washed with 0.5 N NaOH (10 mL) and H₂O (20 mL). The organic fractionwas dried, filtered and concentrated to a brown oil. Flashchromatography (SiO₂; 1/9 EtOAc/CH₂Cl₂, R_(f) 0.35) was attempted, butmaterial crystallized on the column. The silica gel was washed withMeOH. Fractions containing the product (5) (Scheme IV) were concentratedand used without further purification (783 mg, 54.3%): ¹H NMR (200 MHz,CDCl₃) δ 8.24 (m, 2H, Ar—H), 7.45 (m, 3H, Ar—H), 5.24 (br s, 2H, NH2),3.98 (s, 4H, 2×acetal-CH₂), 3.60-3.15 (m, 2H, CH₂), 1.38 (s, 3H, CH₃);MS (ES): 288.1 (M⁺+1).

[0326] Preparation of compound (20) (Scheme VIII): A solution of acetal(19) (4.43 g, 20.6 mmol)¹, benzamine hydrochloride (3.22 g, 20.6 mmol),and DBU (6.15 mL, 41.2 mmol) in dry DMF (20 mL) was heated to 85° C. forfifteen hours. The mixture was diluted with 100 mL of CHCl₃, and washedwith H₂O (2×50 mL). The organic fraction was dried, filtered, andconcentrated to a dark brown oil. The dark brown oil was stirred in 1NHCl (100 mL) for 2 hours at room temperature. The resulting slurry wasfiltered yielding the HCl salt of (20) as a tan solid (3.60 g, 70.6%);1H NMR (200 MHz, DMSO-d6) 11.92 (s 1H), 8.05 (m, 2H, Ar—H), 7.45 (m, 3H,Ar—H), 7.05 (s, 1H, pyrrole-H); MS (ES): 212.1 (M⁺+1).

[0327] Preparation 4:

[0328] A solution of acetal (5) (700 mg, 2.44 mmol) in 1 N HCl (40 mL)was stirred for 2 h at RT. The resultant slurry was filtered yieldingthe HCl salt of 2-phenyl-6-methyl-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one asa tan solid (498 mg, 78.0%): ¹H NMR (200 MHz, DMSO-d₆) δ 11.78 (s, 1H),8.05 (m, 2H, Ar—H), 7.45 (m, 3H, Ar—H), 6.17 (s, 1H, pyrrole-H), 2.25(s, 3H, CH₃); MS (ES): 226.1 (M⁺+1).

[0329] Preparation 5:

[0330] A modification of the Chen et al. cyclization method was used.¹To an ice-cooled (0° C.) solution of bromide (9), (Scheme V; 20.0 g, 108mmol; 90% pure) in isopropyl alcohol (60 mL) was slowly added a solutionof α-methylbenzylamine (12.5 mL, 97.3 mmol). The black solution wasallowed to warm to RT and stir for 15 h. The mixture was diluted withEtOAc (200 mL) and washed with 0.5 N NaOH (50 mL). The organic fractionwas dried, filtered, and concentrated to a black tar (19.2 g; 94%). Theresidue was partially purified by flash chromatography (SiO₂; 4/96MeOH/CH₂Cl₂, R_(f) 0.35) to a black solid (6.38 g, 31%) as the compounddl-1-(1-phenylethyl)-2-amino-3-cyano-4-methylpyrrole: MS (ES): 226.1(M⁺+1).

[0331] Preparation 6:

[0332] To a solution ofdl-1-(1-phenylethyl)-2-amino-3-cyano-4,5-dimethylpyrrole¹ (14.9 g, 62.5mmol) and pyridine (10.0 mL) in dichloromethane (50.0 mL) was addedbenzoyl chloride (9.37 g, 66.7 mmol) at 0° C. After stirring at 0° C.for 1 hr, hexane (10.0 mL) was added to help precipitation of product.Solvent was removed in vacuo and the solid was recrystallized fromEtOH/H₂O to give 13.9 g (65%) ofdl-1-(1-phenylethyl)-2-phenylcarbonylamino-3-cyano-4,5-dimethylpyrrole.mp 218-221° C.; ¹H NMR (200 MHz, CDCl₃) δ 1.72 (s, 3H), 1.76 (d, J=7.3Hz, 3H), 1.98 (s, 3H), 5.52 (q, J=7.3 Hz, 1H), 7.14-7.54 (m, 9H),7.68-7.72 (dd, J=1.4 Hz, 6.9 Hz , 2H), 10.73 (s, 1H); MS (ES): 344.4(M⁺+1).

[0333] The following compounds were obtained in a similar manner.

[0334] Preparation 6A:

[0335]dl-1-(1-phenylethyl)-2-(3-pyridyl)carbonylamino-3-cyano-4,5-dimethylpyrrole.¹H NMR (200 MHz, CDCl₃) δ 1.83 (d, J=6.8 Hz, 3H), 2.02 (s, 3H), 2.12 (s,3H), 5.50 (q, J=6.8 Hz, 1H), 7.14-7.42 (m, SH), 8.08 (m, 2H), 8.75 (m,3H); MS (ES): 345.2 (M⁺+1).

[0336]dl-1-(1-phenylethyl)-2-(2-furyl)carbonylamino-3-cyano-4,5-dimethylpyrrole.¹H NMR (200 MHz, CDCl₃) δ 1.84 (d, J=7.4 Hz, 3H), 1.92 (s, 3H), 2.09 (s,3H), 5.49 (q, J=7.4 Hz, 1H), 6.54 (dd, J=1.8 Hz, 3.6 Hz, 1H), 7.12-7.47(m, 7H); MS (ES): 334.2 (M⁺+1), 230.1.

[0337]dl-1-(1-phenylethyl)-2-(3-furyl)carbonylamino-3-cyano-4,5-dimethylpyrrole.¹H NMR (200 MHz, CDCl₃) δ 1.80 (d, J=7 Hz 3H), 1.89 (s, 3H), 2.05 (s,3H), 5.48 (q, J=7 Hz, 1H), 6.59 (S, 1H), 7.12-7.40 (m, 6H), 7.93 (s,1H); MS (ES) 334.1 (M⁺+1), 230.0.

[0338]dl-1-(1-phenylethyl)-2-cyclopentylcarbonylamino-3-cyano-4,5-dimethylpyrrole.¹H NMR (200 MHz, CDCl₃) δ 1.82 (d, J=7.4 Hz, 3H), 1,88 (s, 3H), 2.05 (s,3H), 1.63-1.85 (m, 8H), 2.63 (m, 1H), 5.43 (q, J=7.4 Hz, 1H), 6.52 (s,1H), 7.05-7.20 (m, 5H); MS (ES): 336.3 (M⁺+1).

[0339]dl-1-(1-phenylethyl)-2-(2-thieyl)carbonylamino-3-cyano-4,5-dimethylpyrrole,¹H NMR (200 MHz, CDCl₃) δ 1.82 (d, J=6.8 Hz, 3H), 1.96 (s, 3H), 2.09 (s,3H), 5.49 (q, J=6.8 Hz, 1H), 7.05-7.55 (m, 8H); MS (ES): 350.1 (M⁺+1),246.0.

[0340]dl-1-(1-phenylethyl)-2-(3-thienyl)carbonylamino-3-cyano-4,5-dimethylpyrrole.

[0341]¹H NMR (200 MHz, CDCl₃) δ 1.83 (d, J=7.0 Hz, 3H), 1.99 (s, 3H),2.12 (s, 3H), 5.49 (q, J=7.0 Hz, 1H), 6.90 (m, 1H), 7.18-7.36 (m, 6H),7.79 (m, 1H); MS (ES): 350.2 (M⁺+1), 246.1.

[0342]dl-1-(1-phenylethyl)-2-(4-fluorophenyl)carbonylamino-3-cyano-4,5-dimethylpyrrole.

[0343]¹H NMR (200 MHz, CDCl₃) δ 1.83 (d, J=7.4 Hz, 3H), 1.96 (s, 3H),2.08 (s, 3H), 5.51 (q, J=7.4 Hz, 1H), 7.16-7.55 (m, 9H); MS (ES): 362.2(M+1), 258.1.

[0344]dl-1-(1-phenylethyl)-2-(3-fluorophenyl)carbonylamino-3-cyano-4,5-dimethylpyrrole.

[0345]¹H NMR (200 MHz, CDCl₃) δ 1.83 (d, J=7.4 Hz 3H), 1.97 (s, 3H),2.10(s, 3H), 5.50 (q, J=7.4 Hz, 1H), 7.05-7.38 (m, 7 H), 7.67-7.74 (m,2H); MS (ES): 362.2 (M⁺+1), 258.1.

[0346]dl-1-(1-phenylethyl)-2-(2-fluorophenyl)carbonylamino-3-cyano-4,5-dimethylpyrrole.¹H NMR (200 MHz, CDCl₃) δ 1.85 (d, J=7.2 Hz, 3H), 1.94 (s, 3H), 2.11 (s,3H), 5.50 (q, J=7.2 hz, 1H), 7.12-7.35 (m, 6H), 7.53 (m, 1H), 7.77 (m,1H), 8.13 (m, 1H); MS (ES): 362.2(M⁺+1), 258.0.

[0347]dl-1-(1-phenylethyl)-2-isoproylcarbonylamino-3-cyano-4,5-dimethylpyrrole.¹H NMR (200 MHz, CDCl₃) δ 1.19 (d, J=7.0 Hz, 6H), 1.82(d, J=7.2 Hz, 3H),1.88 (s, 3H), 2.06 (s, 3H), 2.46 (m, 1H), 5.39 (m, J=7.2 Hz, 1H), 6.64(s, 1H), 7.11-7.36 (m, 5H); MS (ES): 310.2 (M+1), 206.1.

[0348] In the case of acylation ofdl-1-(1-phenylethyl)-2-amino-3-cyano-4-methylpyrrole, monoacylateddl-1-(1-phenylethyl)-2-benzoylamino-3-cyano-4-dimethylpyrrole anddiacylated pyrroledl-1-(1-phenylethyl)-2-dibenzoylamino-3-cyano-4-methylpyrrole wereobtained. Monoacylated pyrrole: ¹H NMR (200 MHz, CDCl₃) δ 7.69 (d, 2H,J=7.8 Hz, Ar—H), 7.58-7.12 (m, 8H, Ar—H), 6.18 (s, 1H, pyrrole-H), 5.52(q, 1H, J=7.2 Hz, CH—CH₃), 2.05 (s, 3H, pyrrole-CH₃), 1.85 (d, 3H, J=7.2Hz, CH—CH ₃); MS (ES): 330.2 (M⁺+1); Diacylated pyrrole: ¹H NMR (200MHz, CDCl₃) δ 7.85 (d, 2H, J=7.7 Hz, Ar—H), 7.74 (d, 2H, J=7.8 Hz,Ar—H), 7.52-7.20 (m, 9H, Ar—H), 7.04 (m, 2H, Ar—H), 6.21 (s, 1H,pyrrole-H), 5.52 (q, 1H, J=7.2 Hz, CH—CH₃), 1.77 (d, 3H, J=7.2 Hz, CH—CH₃), 1.74 (s, 3H, pyrrole-CH₃); MS (ES): 434.1 (M⁺+1).

[0349] Preparation 7:

[0350] To a solution ofdl-1-(1-phenylethyl)-2-phenylcarboxyamido-3-cyano-4,5-dimethylpyrrole(1.0 g, 2.92 mmol) in methanol (10.0 mL) was added concentrated sulfuricacid (1.0 mL) at 0° C. The resulted mixture was refluxed for 15 hr andcooled down to room temperature. The precipitate was filtered to give0.48 g (48%) ofdl-5,6-dimethyl-2-phenyl-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyrimidin-4(3H)-one.¹H NMR (200 MHz, CDCl₃) δ 2.02 (d, J=7.4 Hz, 3H), 2.04 (s, 3H), 2.41 (s,3H), 6.25 (q, J=7.4 Hz, 1H), 7.22-7.50 (m, 9H), 8.07-8.12 (dd, J=3.4 Hz,6.8 Hz, 2H), 10.51 (s, 1H); MS (ES): 344.2 (M⁺+1).

[0351] The following compounds were obtained in a similar manner as thatof Preparation 7:

[0352] dl-5,6-dimethyl-2-(3-pyridyl)-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyrimidin-4(3H)-one. ¹H NMR (200 MHz, CDCl₃) δ 2.03 (d,J=7.2 Hz, 3H), 2.08 (s, 3H), 2.42 (s, 3H), 6.24 (q, J=7.2 Hz, 1H),7.09-7.42 (m, 5H), 8.48 (m, 2H), 8.70 (m, 3H); MS (ES): 345.1 (M⁺+1).

[0353] dl-5,6-dimethyl-2-(2-furyl)-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyrimidin-4(3H)-one. ¹H NMR (200 MHz, CDCl₃) δ 1.98 (d,J=7.8 Hz, 3H), 1.99 (s, 3H), 2.37 (s, 3H), 6.12 (q, J=7.8 Hz, 1H), 6.48(dd, J=1.8 Hz, 3.6 Hz, 1H), 7.17-7.55 (m, 7H), 9.6 (s, 1H); MS (ES):334.2 (M⁺+1).

[0354] dl-5,6-dimethyl-2-(3-furyl)-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyrimidin-4(3H)-one. ¹H NMR (200 MHz, CDCl₃) δ 1.99 (d, J=7 Hz,3H), 2.02 (s, 3H), 2.42 (s, 3H), 6.24 (q, J=7 Hz, 1H), 7.09 (s, 1H),7.18-7.32 (m, SH), 7.48 (s, 1H), 8.51 (s, 1H); MS (ES): 334.2 (M⁺+1).

[0355] dl-5,6-dimethyl-2-cyclopentyl-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyrimidin-4(3H)-one. ¹H NMR (200 MHz, CDCl₃) δ 1.95 (d,J=7.4 Hz, 3H), 2.00 (s, 3H), 2.33 (s, 3H), 1.68-1.88 (m, 8H), 2.97 (m,1H), 6.10 (q, J=7.4 Hz, 1H), 7.16-7.30 (m, 5H), 9.29 (s, 1H); MS (ES):336.3 (M⁺+1).

[0356] dl-5,6-dimethyl-2-(2-thienyl)-7H-7-(1-phenylethyl) pyrrolo[2,3d]pyrimidin-4(3H)-one. ¹H NMR (200 MHz, CDCl₃) δ 2.02 (d, J=7.2 Hz,3H), 2.06 (s, 3H), 2.41 (s, 3H), 6.13 (q, J=7.2 Hz, 1H), 7.12 (dd,J=4.8, 2.8 Hz, 1H), 7.26-7.32 (m, 5H), 7.44 (d, J=4.8 Hz, 1H), 8.01 (d,J=2.8 Hz, 1H) 11.25 (s, 1H); MS (ES) 350.2 (M⁺+1).

[0357] dl-5,6-dimethyl-2-(3-thienyl)-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyrimidin-4(3H)-one. ¹H NMR (200 MHz, CDCl₃) δ 2.00 (d,J=7.4 Hz, 3H), 2.05 (s, 3H), 2.43 (s, 3H), 6.24(q, J=7.4 Hz, 1H),7.24-7.33 (m, 5H), 7.33-7.39 (m, 1H), 7.85 (m, 1H), 8.47 (m, 1H), 12.01(s, 1H); MS (ES): 350.2 (M⁺+1).

[0358] dl-5,6-dimethyl-2-(4-fluorophenyl)-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyrimidin-4(3H)-one. ¹H NMR (200 MHz, CDCl₃) δ 2.01 (d,J=6.8 Hz, 3H), 2.05 (s, 3H), 2.42 (s, 3H), 6.26 (q, J=6.8 Hz, 1H),7.12-7.36 (m, 7H), 8.23-8.30 (m, 2H), 11.82 (s, 1H); MS (ES): 362.3(M⁺+1).

[0359] dl-5,6-dimethyl-2-(3-fluorophenyl)-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyrimidin-4(3H)-one. ¹H NMR (200 MHz, CDCl₃)δ 2.02 (d,J=7.4 Hz, 3H), 2.06 (s, 3H), 2.44 (s, 3H), 6.29 (q, J=7.4 Hz, 1H),7.13-7.51(m, 7H), 8.00-8.04 (m, 2H), 11.72 (s, 1H); MS (ES): 362.2(M⁺+1).

[0360] dl-5,6-dimethyl-2-(2-fluorophenyl)-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyrimidin-4(3H)-one. ¹H NMR (200 MHz, CDCl₃) δ 2.00(d,J=7.2 Hz, 3H), 2.05 (s, 3H), 2.38 (s, 3H), 6.24 (q, J=7.2 Hz, 1H),7.18-7.45 (m, 8H), 8.21 (m, 1H), 9.54 (s, 1H); MS (ES): 362.2 (M⁺+1).

[0361] dl-5,6-dimethyl-2-isopropyl-7H-7-(1-phenylethyl) pyrrolo[2,3d]pyrimidin-4(3H)-one.

[0362]¹H NMR (200 MHz, CDCl₃) δ 1.30 (d, J=6.8 Hz, 3H), 1.32 (d, J=7.0Hz, 3H), 2.01 (s, 3H), 2.34 (s, 3H), 2.90 (m, 1H) 6.13 (m, 1H),7.17-7.34 (m, 5H), 10.16 (s, 1H); MS (ES) 310.2 (M+1).

[0363] Preparation 8:

[0364] A solution ofdl-1-(1-phenylethyl)-2-benzoylamino-3-cyano-4-dimethylpyrrole (785 mg,2.38 mmol) with concentrated H₂SO₄ (1 mL) in DMF (13 mL) was stirred at130° C. for 48 hrs. The black solution was diluted with CHCl₃ (100 mL)and washed with 1 N NaOH (30 mL), and brine (30 mL). The organicfraction was dried, filtered, concentrated, and purified by flashchromatography (SiO₂; 8/2 EtOAc/Hex, R_(f) 0.35) to a brown solid (184mg, 24%) asdl-5-methyl-2-phenyl-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyrimidin-4(3H)-one.¹H NMR (200 MHz, CDCl₃) δ 8.18 (m, 2H, Ar—H), 7.62-7.44 (m, 3H, Ar—H),7.40-7.18 (m, 5H, Ar—H), 6.48 (s, 1H, pyrrole-H), 6.28 (q, 1H, J=7.2 Hz,CH—CH₃), 2.18 (s, 3H, pyrrole-CH₃), 2.07 (d, 3H, J=7.2 Hz, CH—CH ₃); MS(ES): 330.2 (M⁺+1).

[0365] Preparation 9:

[0366] A mixture ofdl-1-(1-phenylethyl)-2-amino-3-cyano-4,5-dimethylpyrrole (9.60 g, 40.0mmol) and of formic acid (50.0 mL, 98%) was refluxed for 5 hr. Aftercooling down to room temperature and scratching the sides of flask,copious precipitate was formed and filtered. The material was washedwith water until washings showed neutral pH to givedl-5,6-dimethyl-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyrimidin-4 (3H)-one.¹H NMR (200 MHz, CDCl₃) δ 1.96 (d, J=7.4 hz, 3H), 2.00 (s, 3H), 2.38 (s,3H), 6.21 (q, J=7.4 Hz, 1H), 7.11-7.35 (m, 5H), 7.81 (s, 1H), 11.71 (s,1H); MS (ES): 268.2 (M⁺+1).

[0367] Preparation 10:

[0368] dl-5,6-dimethyl-2-phenyl-7H-7-(1-phenylethyl) pyrrolo[2,3d]pyrimidin-4(3H)-one (1.0 g, 2.91 mmol) was suspended in polyphosphoricacid (30.0 mL). The mixture was heated at 100° C. for 4 hrs. The hotsuspension was poured onto ice water, stirred vigorously to dispersesuspension, and basified to pH 6 with solid KOH. The resulting solid wasfiltered and collected to give 0.49 g (69%) of5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one. ¹H NMR (200MHz, DMSO-d₆) δ 2.17 (s, 3H), 2.22 (s, 3H), 7.45 (br, 3H), 8.07 (br,2H,), 11.49 (s, 1H), 11.82 (s, 1H); MS (ES) 344.2 (M⁺+1).

[0369] The following compounds were obtained in a similar manner as thatof Preparation 10:

[0370] 5-methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one. MS (ES):226.0 (M⁺+1).

[0371] 5,6-dimethyl-2-(3-pyridyl)-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one.MS (ES): 241.1 (M⁺+1).

[0372] 5,6-dimethyl-2-(2-furyl)-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one. ¹HNMR (200 MHz, DMSO-d₆) δ 2.13 (s, 3H), 2.18 (s, 3H), 6.39 (dd, J=1.8,3.6 Hz, 1H), 6.65 (dd, J=1.8 Hz, 3.6 Hz, 1H), 7.85 (dd, J=1.8, 3.6 Hz,1H,), 11.45 (s, 1H), 11.60 (s, 1H); MS (ES): 230.1 (M⁺+1).

[0373] 5,6-dimethyl-2-(3-furyl)-7H-pyrrolo[2,3d]pyrimidin-4 (3H)-one. ¹HNMR (200 MHz, DMSO-d₆) δ 2.14 (s, 3H), 2.19 (s, 3H), 6.66 (s, 1H), 7.78(s, 1H), 8.35 (s, 1H), 11.3 (s, 1H), 11.4 (s, 1H); MS (ES): 230.1(M⁺+1).

[0374] 5,6-dimethyl-2-cyclopentyl-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one.¹H NMR (200 MHz, DMSO-d₆) δ 1.57-1.91 (m, 8H), 2.12 (s, 3H), 2.16 (s,3H), 2.99 (m, 1H), 11.24 (s, 1H), 11.38 (s, 1H); MS (ES): 232.2 (M⁺+1).

[0375] 5,6-dimethyl-2-(2-thienyl)-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one.¹H NMR (200 MHz, DMSO-d₆) δ 2.14 (s, 3H), 2.19 (s, 3H), 7.14 (dd, J=3.0,5.2 Hz, 1H), 7.70 (d, J=5.2 Hz 1H), 8.10 (d, J=3.0 Hz, 1H), 11.50 (s,1H); MS (ES): 246.1 (M⁺+1).

[0376] 5,6-dimethyl-2-(3-thienyl)-7H-pyrrolo[2,3d]pyrimidin-4 (3H)-one.¹H NMR (200 MHz, DMSO-d₆) δ 2.17 (s, 3H), 2.21(s, 3H), 7.66(m, 1H), 7.75(m, 1H), 8.43 (m, 1H), 11.47 (s, 1H), 11.69 (s, 1H); MS (ES): 246.1(M⁺+1).

[0377]5,6-dimethyl-2-(4-fluorophenyl)-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one. ¹HNMR (200 MHz, DMSO-d₆) δ 2.17 (s, 3H), 2.21 (s, 3H), 7.31 (m, 2H), 8.12(m, 2H), 11.47 (s, 1H); MS (ES) 258.2 (M⁺+1).

[0378]5,6-dimethyl-2-(3-fluorophenyl)-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one. ¹HNMR (200 MHz, DMSO-d₆) δ 2.18 (s, 3H), 2.21 (s, 3H), 7.33 (m, 1H), 7.52(m, 1H), 7.85-7.95 (m, 2H), 11.56 (s, 1H), 11.80 (s, 1H); MS (ES): 258.1(M⁺+1).

[0379]5,6-dimethyl-2-(2-fluorophenyl)-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one. ¹HNMR (200 MHz, DMSO-d₆) δ 2.18 (s, 3H), 2.22 (s, 3H), 7.27-7.37 (m, 2H),7.53 (m 1H), 7.68 (mi ,1H), 11.54 (s, 1H), 11.78 (s, 1H); MS (ES): 258.1(M⁺+1).

[0380] 5,6-dimethyl-2-isopropyl-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one. ¹HNMR (200 MHz, DMSO-d₆) δ 1.17 (d, J=6.6 Hz, 6H), 2.11 (s, 3H), 2.15 (s,3H), 2.81 (m, 1H), 11.20 (s, 1H), 11.39 (s, 1H); MS (ES): 206.1 (M⁺+1).

[0381] 5,6-dimethyl-7H-pyrrolo[2,3d]pyrimidin-4 (3H)-one. ¹H NMR (200MHz, DMSO-d₆) δ 2.13 (s, 3H), 2.17 (s, 3H), 7.65 (s, 1H); MS (ES): 164.0(M⁺+1).

[0382] Preparation 11:

[0383] A solution of5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidin-4(3H)-one (1.0 g, 4.2mmol) in phosphorus oxychloride (25.0 mL) was refluxed for 6 hr and thenconcentrated in vacuo to dryness. Water was added to the residue toinduce crystallization and the resulting solid was filtered andcollected to give 0.90 g (83%) of4-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. ¹H NMR (200MHz, DMSO-d₆) δ_(—)2.33 (s, 3H), 2.33 (s, 3H), 7.46-7.49 (m, 3H),8.30-8.35 (m, 2H), 12.20 (s, 1H); MS (ES): 258.1 (M⁺+1).

[0384] The following compounds were obtained in a similar manner as thatof Preparation 11:

[0385] 4-chloro-5-methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS (ES):244.0 (M⁺+1).

[0386] 4-chloro-6-methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS (ES)244.0 (M⁺+1).

[0387] 4-chloro-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. ¹H NMR (200 MHz,DMSO-d6) 8.35 (2, 2H), 7.63 (br s, 1H), 7.45 (m, 3H), 6.47 (br s, 1H);MS (ES): 230.0 (M⁺+1).

[0388] 4-chloro-5,6-dimethyl-2-(3-pyridyl)-7H-pyrrolo[2,3d]pyrimidine.MS (ES): 259.0 (M⁺+1).

[0389] 4-chloro-5,6-dimethyl-2-(2-furyl)-7H-pyrrolo [2,3d] pyrimidine.¹H NMR (200 MHz, DMSO-d₆) δ 2.35 (s, 3H), 2.35 (s, 3H), 6.68 (dd, J=1.8,3.6 Hz, 1H), 7.34 (dd, J=1.8 Hz, 3.6 Hz, 1H), 7.89 (dd, J=1.8, 3.6 Hz,1H); MS (ES): 248.0 (M⁺+1).

[0390] 4-chloro-5,6-dimethyl-2-(3-furyl)-7H-pyrrolo[2,3d]pyrimidine. ¹HNMR (200 MHz, DMSO-d₆) δ 2.31 (s, 3H), 2.31 (s, 3H), 6.62 (s, 1H), 7.78(s, 1H), 8.18 (s, 1H), 12.02 (s, 1H); MS (ES): 248.1 (M⁺+1).

[0391] 4-chloro-5,6-dimethyl-2-cyclopentyl-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, DMSO-d₆) δ 1.61-1.96 (m, 8H), 2.27 (s, 3H), 2.27 (s,3H), 3.22 (m, 1H), 11.97 (s, 1H); MS (ES): 250.1 (M⁺+1).

[0392] 4-chloro-5,6-dimethyl-2-(2-thienyl)-7H-pyrrolo[2,3d] pyrimidine.¹H NMR (200 MHz, DMSO-d₆) δ 2.29 (s, 3H), 2.31 (s, 3H), 7.14 (dd, J=3.1Hz, 4.0 Hz, 1H), 7.33 (d, J=4.9 Hz, 1H), 7.82 (d, J=3.1 Hz, 1H), 12.19(s, 1H); MS (ES) 264.1 (M⁺+1).

[0393] 4-chloro-5,6-dimethyl-2-(3-thienyl)-7H-pyrrolo[2,3d] pyrimidine.¹H NMR (200 MHz, DMSO-d₆) δ 2.32 (s, 3H), 2.32 (s, 3H), 7.62 (dd, J=3.0,5.2 Hz, 1H), 7.75 (d, J=5.2 Hz, 1H), 8.20 (d, J=3.0 Hz, 1H); MS (ES)264.0 (M⁺+1).

[0394] 4-chloro-5,6-dimethyl-2-(4-fluorophenyl)-7H-pyrrolo[2,3d]pyrimidine. ¹H NMR (200 MHz, DMSO-d₆) δ 2.33(s, 3H), 2.33 (s, 3H), 7.30(m, 2H), 8.34 (m, 2H), 12.11 (s, 1H); MS (ES) 276.1. (M⁺+1).

[0395]4-chloro-5,6-dimethyl-2-(3-fluorophenyl)-7H-pyrrolo[2,3d]pyrimidine. ¹HNMR (200 MHz, DMSO-d₆) δ 2.31(s, 3H), 2.33 (s, 3H), 7.29(m, 1H), 7.52(m, 1H), 7.96 (m, 1H), 8.14(m, 1H), 11.57 (s, 1H); MS (ES): 276.1(M⁺+1).

[0396] 4-chloro-5,6-dimethyl-2-(2-fluorophenyl)-7H-pyrrolo[2,3d]pyrimidine. ¹H NMR (200 MHz, DMSO-d₆) δ 2.34 (s, 3H), 2.34 (s, 3H), 7.33(m, 2H), 7.44 (m, 1H), 7.99 (m, 1H), 12.23 (s, 1H); MS (ES): 276.1(M⁺+1).

[0397] 4-chloro-5,6-dimethyl-2-isopropyl-7H-pyrrolo[2,3d]pyrimidine. ¹HNMR (200 MHz, DMSO-d₆) δ 1.24 (d, J=6.6 Hz, 6H), 2.28 (s, 3H), 2.28 (s,3H), 3.08 (q, J=6.6 Hz, 1H), 11.95 (s, 1H); MS (ES): 224.0 (M⁺+1).

[0398] 4-chloro-5,6-dimethyl-7H-pyrrolo[2,3d]pyrimidine. ¹H NMR (200MHz, DMSO-d₆) δ 2.31 (s, 3H), 2.32 (s, 3H), 8.40 (s, 1H); MS (ES): 182.0(M⁺+1).

[0399] dl-4-chloro-5,6-dimethyl-2-phenyl-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyrimidine.

[0400] Preparation 12:

[0401] To a solution of dl-1,2-diaminopropane (1.48 g, 20.0 mmol) andsodium carbonate (2.73 g, 22.0 mmol) in dioxane (100.0 mL) and water(100.0 mL) was added di-tert-dicarbonate (4.80 g, 22.0 mmol) at roomtemperature. The resulted mixture was stirred for 14 hr. Dioxane wasremoved in vacuo. The precipitate was filtered off and the filtrate wasconcentrated in vacuo to dryness. The residue was triturated with EtOAcand then filtered. The filtrate was concentrated in vacuo to dryness togive a mixture of dl-1-amino-2-(1,1-dimethylethoxy)carbonylamino-propaneand dl-2-amino-1-(1,1-dimethylethoxy)carbonylamino-propane which werenot separable by normal chromatography method. The mixture was used forthe reaction in Example 8.

[0402] Preparation 13:

[0403] To solution of Fmoc-β-Ala-OH (1.0 g, 3.212 mmol) and oxalylchloride (0.428 g, 0.29 mL, 3.373 mmol) in dichloromethane (20.0 mL) wasadded a few drops of N,N-dimethylformamide at 0° C. The mixture wasstirred at room temperature for 1 hr followed by addition ofcyclopropylmethylamine (0.229 g, 0.28 mL, 3.212 mmol) and triethylamine(0.65 g, 0.90 mL, 6.424 mmol). After 10 min, the mixture was treatedwith 1 M hydrochloride (10.0 mL) and the aqueous mixture was extractedwith dichloromethane (3×30.0 mL). The organic solution was concentratedin vacuo to dryness. The residue was treated with a solution of 20%piperidine in N,N-dimethylforamide (20.0 mL) for 0.5 hr. After removalof the solvent in vacuo, the residue was treated with 1 M hydrochloride(20.0 mL) and ethyl acetate (20.0 mL). The mixture was separated and theaqueous layer was basified with solid sodium hydroxide to pH=8. Theprecipitate was removed by filtration and the aqueous solution wassubjected to ion exchange column eluted with 20% pyridine to give 0.262g (57a) of N-cyclopropylmethyl β-alanine amide. ¹H NMR (200 MHz, CD₃OD)δ_(—)0.22 (m, 2H), 0.49 (m, 2H), 0.96 (m, 2H), 2.40 (t, 2H) 2.92 (t,2H), 3.05 (d, 2H); MS (ES): 143.1 (M⁺+1).

[0404] Preparation 14:

[0405] N-tert-butoxycarbonyl- trans-1,4-cyclohexyldiamine.trans-1,4-cyclonexyldiamine (6.08 g, 53.2 mmol) was dissolved indichloromethane (100 mL). A solution of di-t-butyldicarbonate (2.32 g,10.65 mmol in 40 mL dichloromethane) was added via cannula. After 20hours, the reaction was partitioned between CHCl₃ and water. The layerswere separated and the aqueous layer was extracted with CHCl₃ (3×). Thecombined organic layers were dried over MgSO₄, filtered and concentratedto yield 1.20 g of a white solid (53%). ¹H-NMR (200 MHz, CDC₃): δ1.0-1.3 (m, 4H), 1.44 (s, 9H), 1.8-2.1 (m, 4H), 2.62 (brm, 1H), 3.40(brs, 1H), 4.37 (brs, 1H0; MS (ES): 215.2 (M⁺+1).

[0406] 4-(N-acetyl)-N-tert-butoxycarbonyl-trans-1,4-cyclohexyl diamine.

[0407] N-tert-butoxycarbonyl-trans-1,4-cyclohexyldiamine (530 mg, 2.47mmol) was dissolved in dichloromethane (20 mL). Acetic anhydride (250mg, 2.60 mmol) was added dropwise. After 16 hours, the reaction wasdiluted with water and CHCl₃. The layers were separated and the aqueouslayer was extracted with CHCl₃ (3×). The combined organic layers weredried over MgSO₄, filtered and concentrated. Recrystallization(EtOH/H₂O) yielded 190 mg of white crystals (30%). ¹H NMR (200 MHz,CDCl₃): δ 0.9-1.30 (m, 4H), 1.43 (s, 9H), 1.96-2.10 (m, 7H), 3.40 (brs,1H), 3.70 (brs, 1H), 4.40 (brs, 1H), 4.40 (brs, 1H); MS (ES): 257.2(M⁺+1), 242.1 (M⁺−15), 201.1 (M⁺−56).

[0408]4-(4-trans-acetamidocyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-(1-phenylethyl)pyrrolo[2,3d]pyrimidine.4-(N-acetyl)-N-tert-butoxycarbonyl-trans-1,4-cyclohexyldiamine (190 mg,0.74 mmol), was dissolved in dichloromethane (5 mL) and diluted with TFA(6 ml). After 16 hours, the reaction was concentrated. The crude solid,DMSO (2 mL), NaHCO₃ (200 mg, 2.2 mmol) and4-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine (35 mg, 0.14mmol) were combined in a flask and heated to 130° C. After 4.5 hours,the reaction was cooled to room temperature and diluted with EtOAc andwater. The layers were separated and the aqueous layer was extractedwith EtOAc (3×). The combined organic layers were dried over MgSO₄,filtered and concentrated. Chromatography (silica preparatory plate;20:1 CHCl₃:EtOH) yielded 0.3 mg of a tan solid (1% yield). MS (ES):378.2 (M⁺+1).

[0409]4-(N-methanesulfonyl)-N-tert-butoxycarbonyl-trans-1,4-cyclohexyldiamine.

[0410] trans-1,4-cyclohexyldiamine (530 mg, 2.47 mmol) was dissolved indichloromethane (20 ml) and diluted with pyridine (233 mg, 3.0 mmol).Methanesulfonyl chloride (300 mg, 2.60 mmol) was added dropwise. After16 hours, the reaction was diluted with water and CHCl₃. The layers wereseparated and the aqueous layer was extracted with CHCl₃ (3×). Thecombined organic layers were dried over MgSO₄, filtered andconcentrated. recrystallization (EtOH/H₂O) yielded 206 mg of whitecrystals (29%). ¹H-NMR (200 MHz, CDCl₃): δ 1.10-1.40 (m, 4H), 1.45 (s,9H), 2.00-2.20 (m, 4H), 2.98 (s, 3H), 3.20-3.50 (brs, 2H), 4.37 (brs,1H); MS (ES) 293.1 (M⁺+1), 278.1 (M⁺−15), 237.1 (M⁺−56).

[0411]4-(4-trans-methanesulfamidocyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-(1-phenylethyl)pyrrolo[2,3d]pyrimidine.

[0412] 4-(N-sulfonyl)-N-tert-butoxycarbonyl-trans-1,4-cyclohexyldiamine(206 mg, 0.71 mmol), was dissolved in dichloromethane (5 ml) and dilutedwith TFA (6 ml). After 16 hours, the reaction was concentrated. Thecrude reaction mixture, DMSO (2 ml), NaHCO₃ (100 mg, 1.1 mmol) and1-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine were combinedin a flask and heated to 130° C. After 15 hours, the reaction was cooledto room temperature, and diluted with EtOAc (3×). The combined organiclayers were dried over MgSO₄, filtered and concentrated. Chromatography(silica preparatory plate, 20:1 CHCl₃/EtOH) yielded 2.6 mg of a tansolid (5- yield). MS (ES): 414.2 (M⁺+1).

EXAMPLE 1

[0413] A solution of 4-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine (0.50 g, 1.94 mmol) and 4-trans-hydroxy cyclohexylamine (2.23g, 19.4 mmol) in methyl sulfoxide (10.0 mL) was heated at 130° C. for 5hr. After cooling down to room temperature, water (10.0 mL) was addedand the resulted aqueous solution was extracted with EtOAc (3×10.0 mL).The combined EtOAc solution was dried (MgSO₄) and filtered, the filtratewas concentrated in vacuo to dryness, the residue was chromatographed onsilica gel to give 0.49 g (75%) of4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.mp 197-199° C.; ¹H NMR (200 MHz, CDCl₃) δ_(—)1.25-1.59 (m, 8H), 2.08 (s,3H), 2.29 (s, 3H), 3.68-3.79 (m, 1H), 4.32-4.38 (m, 1H), 4.88 (d, J=8Hz, 1H), 7.26-7.49 (m, 3H), 8.40-8.44 (dd, J=2.2, 8 Hz, 2H), 10.60 (s,1H); MS (ES): 337.2 (M⁺+1).

[0414] The following compounds were obtained in a similar manner to thatof Example 1:

[0415]4-(4-trans-hydroxycyclohexyl)amino-6-methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, CDCl₃) δ 11.37 (s, 1H, pyrrole-NH), 8.45 (m, 2H, Ar—H),7.55 (m, 3H, Ar—H), 6.17 (s, 1H, pyrrole-H), 4.90 (br d, 1H, NH), 4.18(m, 1H, CH—O), 3.69 (m, 1H, CH—N), 2.40-2.20 (m, 2H), 2.19-1.98 (m, 2H),2.25 (s, 3H, CH3) 1.68-1.20 (m, 4H); MS (ES): 323.2 (M⁺+1).

[0416]4-(4-trans-hydroxycyclohexyl)amino-5-methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, CDCl₃) δ 11.37 (s, 1H, pyrrole-NH), 8.40 (m, 2H, Ar—H),7.45 (m, 3H, Ar—H), 5.96 (s, 1H, pyrrole-H), 4.90 (br d, 1H, NH), 4.18(m, 1H, CH—O), 3.69 (m, 1H, CH—N), 2.38-2.20 (m, 2H), 2.18-1.98 (m, 2H)2.00 (s, 3H, CH3) 1.68-1.20 (m, 4H); MS (ES): 323.2 (M⁺+1).

[0417] 4-(4-trans-hydroxycyclohexyl)amino-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. mp 245.5-246.5° C.; ¹H NMR (200 MHz, CD₃OD) δ 8.33(m, 2H,Ar—H), 7.42 (m, 3H, Ar—H), 7.02 (d, 1H, J=3.6 Hz, pyrolle-H), 6.53 (d,1H, J=3.6 Hz, pyrolle-H), 4.26 (m, 1H, CH—O), 3.62 (m,1H, CH—N),2.30-2.12 (m, 2H), 2.12-1.96 (m, 2H), 1.64-1.34 (m, 4H); MS, M+1=309.3;Anal (C₁₈H₂₀N₄O) C, H, N.

[0418]4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-(3-pyridyl)-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, CDCl₃) δ_(—)1.21-1.54 (m, 8H); 2.28 (s, 3H); 2.33 (s,3H); 3.70 (m, 1H), 4.31(m, 1H), 4.89 (d, 1H), 7.40 (m, 1H), 8.61 (m,2H), 9.64 (m, 1H); MS (ES): 338.2 (M⁺+1).

[0419]4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-(2-furyl)-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, CDCl₃) δ 1.26-1.64(m, 8H), 2.22 (s, 3H), 2.30 (s, 3H),3.72(m, 1H), 4.23 (m, 1H), 4.85 (d, 1H), 6.52 (m, 1H), 7.12 (m, 1H),7.53 (m, 1H), 9.28 (s, 1H); MS (ES): 327.2 (M⁺+1).

[0420]4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-(3-furyl)-7H-pyrrolo[2,3d]pyrimidine.1H NMR (200 MHz, CDCl₃) δ 1.25-1.63 (m, 8H), 2.11 (s, 3H), 2.27 (s, 3H),3.71(m, 1H), 4.20 (m, 1H), 4.84 (d, 1H), 7.03 (m, 1H), 7.45(m, 1H),8.13(m, 1H), 10.38 (m, 1H); MS (ES): 327.2 (M⁺+1).

[0421]4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-cyclopentyl-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, CDCl₃) δ 1.26-2.04 (m, 16H), 2.26 (s, 3H), 2.27 (s,3H), 3.15(m, 1H), 3.70 (m, 1H), 4.12 (m, 1H), 4.75(d, 1H); MS (ES):329.2 (M⁺+1).

[0422] 4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-(2-thienyl)-7H-pyrrolo[2,3d]pyrimidin-4-amine. ¹H NMR (200 MHz, CDCl₃) δ 1.28-1.59(m, 8H), 2.19 (s, 3H) 2.29 (s, 3H), 3.74 (m, 1H), 4.19 (m, 1H), 4.84 (d,1H), 7.09 (m, 1H), 7.34 (m, 1H), 7.85 (m, 1H), 9.02 (s, 1H); MS (ES):343.2 (M⁺+1).

[0423]4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-(3-thienyl)-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, CDCl₃) δ 1.21-1.60 (m, 8H), 1.98 (s, 3H), 2.23 (s, 3H),3.66 (m, 1H), 4.22 (m, 1H), 7.27 (m, 1H), 7.86 (m, 1H), 8.09 (m, 1H),11.23 (s, 1H); MS (ES): 343.2 (M⁺+1).

[0424]4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-(4-fluorophenyl)-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, CDCl₃) δ 1.26-1.66 (m, 8H), 1.94 (s, 3H), 2.28 (s, 3H),3.73 (m, 1H), 4.33 (m, 1H), 4.92 (d, 1H), 7.13 (m, 2H), 8.41 (m, 2H),11.14 (s, 1H); MS (ES): 355.2 (M⁺+1).

[0425]4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-(3-fluorophenyl)-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, CDCl₃) δ 1.26-1.71 (m, 8H), 2.06 (s, 3H), 2.30 (s, 3H),3.72 (m, 1H), 4.30 (m, 1H), 4.90 (d, 1H), 7.09 (m, 1H), 7.39 (m, 1H),8.05 (m, 1H), 8.20 (m, 1H), 10.04 (s. 1H); MS (ES): 355.2 (M⁺+1).

[0426]4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-(2-fluorophenyl)-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, CDCl₃) δ 1.30-1.64 (m, 8H), 2.17 (s, 3H), 2.31 (s, 3H),3.73 (m, 1H), 4.24 (m, 1H), 4.82 (d, 1H), 7.28 (m, 2H), 8.18 (m, 1H),9.02 (m, 1H), 12.20 (s, 1H); MS (ES): 355.3 (M⁺+1).

[0427]4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-isopropyl-7H-pyrrolo[2,3d]pyrimidine¹H NMR (200 MHz, CDCl₃) δ 1.31 (d, J=7.0 Hz, 6H), 1.30-1.65 (m, 8H),2.27 (s, 3H), 2.28 (s, 3H), 3.01 (m, J=7.0 Hz, 1H), 3.71 (m, 1H), 4.14(m, 1H), 4.78 (d, 1H); MS (ES): 303.2.

[0428]dl-4-(2-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-isopropyl-7H-pyrrolo[2,3d]pyrimidineH NMR (200 MHz, CDCl₃) d 1.31-1.42 (br, 4H), 1.75-1.82 (br, 4H), 2.02(s, 3H), 2.29 (s, 3H), 3.53 (m, 1H), 4.02 (m, 1H), 5.08 (d, 1H),7.41-7.48 (m, 3H), 8.30 (m, 2H), 10.08 (s, 1H); MS (ES): 337.2 (M⁺+1).

[0429]4-(3,4-trans-dihydroxycyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.MS (ES) :353.2 (M⁺+1).

[0430] 4-(3,4-cis-dihydroxylcyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS (ES): 353.2(M +1).

[0431]4-(2-acetylaminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.

[0432] mp 196-199° C.; ¹H NMR (200 MHz, CDCl₃) δ 1.72 (s, 3H), 1.97 (s,3H), 2.31 (s, 3H), 3.59 (m, 2H), 3.96 (m, 2H), 5.63 (br, 1H), 7.44-7.47(m, 3H), 8.36-8.43 (dd, J=1 Hz, 7 Hz, 2H), 10.76 (s, 1H); MS (ES): 324.5(M⁺+1).

[0433]dl-4-(2-trans-hydroxycyclopentyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.¹

[0434]¹H NMR (200 MHz, CDCl₃) δ 1.62 (m, 2H), 1.79 (br, 4H), 1.92 (s,3H), 2.29 (s, 3H), 4.11 (m, 1H), 4.23 (m, 1H), 5.28 (d, 1H), 7.41-7.49(m, 3H), 8.22 (m, 2H), 10.51 (s, 1H); MS (ES): 323.2 (M⁺+1).

[0435]dl-4-(3-trans-hydroxycyclopentyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.¹

[0436]¹H NMR (200 MHz, CDCl₃) δ 1.58-1.90 (br, 6 H, 2.05 (s, 3H), 2.29(s, 3H), 4.48-4.57 (m, 1H), 4.91-5.01 (m, 2H), 7.35-7.46 (m, 3H),8.42-8.47 (m, 2H), 10.11 (s, 1H); MS (ES): 323.2 (M⁺+1).

[0437]dl-4-(3-cis-hydroxycyclopentyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.¹

[0438]¹H NMR (200 MHz, CDCl₃) δ_(—)1.82-2.28 (br, 6H), 2.02 (s, 3H) 2.30(s, 3H), 4.53-4.60 (m, 1H), 4.95-5.08 (m, 1H), 5.85-5.93 (d, 1H),7.35-7.47 (m, 3H), 8.42-8.46 (m, 2H), 10.05 (s, 1H); MS (ES): 323.2(M⁺+1).

[0439] 4-(3,4-trans-dihydroxycyclopentyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.¹ ¹H NMR (200 MHz, CDCl₃) δ_(—)1.92-1.99(br, 2H), 2.14 (s, 3H), 2.20 (br, 2H), 2.30 (s, 3H), 2.41-2.52 (br, 2H),4.35 (m, 2H), 4.98 (m, 2H), 7.38-7.47 (m, 3H), 8.38-8.42 (m, 2H), 9.53(s, 1H); MS (ES): 339.2 (M⁺+1).

[0440]4-(3-amino-3-oxopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.

[0441]¹H NMR (200 MHz, CDCl₃) δ_(—)2.02 (s, 3H), 2.29 (s, 3H), 2.71 (t,2H), 4.18 (m, 2H), 5.75-5.95 (m, 3H), 7.38-7.48 (m, 3H), 8.37-8.41 (m,2H), 10.42 (s, 1H); MS (ES): 310.1 (M⁻+1).

[0442]4-(3-N-cyclopropylmethylamino-3-oxopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, CD₃OD) δ_(—)0.51 (q, 2H), 0.40 (q, 2H), 1.79-1.95 (br,1×), 2.36 (s, 3H), 2.40 (s, 3H), 2.72 (t, 2H), 2.99 (d, 2H), 4.04 (t,2H), 7.58-7.62 (m, 3H), 8.22-8.29 (m, 2H); MS (ES): 364.2 (M⁺+1).

[0443]4-(2-amino-2-oxoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine¹H NMR (200 MHz, CD₃OD) δ 2.31 (s, 3H), 2.38 (s, 3H), 4.26 (s, 2H), 7.36(m, 3H), 8.33 (m, 2H) MS (ES): 396.1 (M⁺+1).

[0444]4-(2-N-methylamino-2-oxoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, CDCl₃) δ 1.99 (s, 3H), 2.17 (s, 3H), 2.82 (d, 3H), 4.39(d, 2H), 5.76 (t, 1H), 6.71 (br, 1H), 7.41-7.48 (m, 3H), 8.40 (m, 2H),10.66 (s, 1H); MS (ES) 310.1 (M⁺+1).

[0445]4-(3-tert-butyloxyl-3-oxopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, CDCl₃) δ_(—)1.45 (s, 9H), 1.96 (s, 3H), 2.29 (s, 3H),2.71 (t, 2H), 4.01 (q, 2H), 5.78 (t, 1H), 7.41-7.48 (m, 3H), 8.22-8.29(m, 2H); MS (ES): 367.2 (M⁺+1).

[0446]4-(2-hydroxyethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, CDCl₃) δ 1.92 (s, 3H), 2.29 (s, 3H), 3.81-3.98 (br,4H), 5.59 (t, 1H), 7.39-7.48 (m, 3H), 8.37 (m, 2H), 10.72 (s, 1H); MS(ES): 283.1 (M⁺+1).

[0447]4-(3-hydroxypropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, CDCl₃) δ 1.84 (m, 2H), 1.99 (s, 3H), 2.32 (s, 3H), 3.62(t, 2H), 3.96 (m, 2H), 3.35 (t, 1H), 7.39-7.48 (m, 3H), 8.36 (m, 2H),10.27 (s, 1H); MS (ES): 297.2 (M+1).

[0448] 4-(4-hydroxybutyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. ¹H NMR (200 MHz, CDCl₃) (1.71-1.82 (m, 4H), 1.99 (s,3H), 2.31 (s, 3H), 3.68-3.80 (m, 4H), 5.20 (t, 1H), 7.41-7.49 (m, 3H),8.41(m, 2H), 10.37 (s, 1H); MS (ES): 311.2 (M⁺+1).

[0449]4-(4-trans-acetylaminocyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.

[0450]4-(4-trans-methylsulfonylaminocyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.

[0451]4-(2-acetylaminoethyl)amino-5,6-dimethyl-2-phenyl-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyrimidine.

[0452]4-(4-trans-hydoxycyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-1-phenylethyl)pyrrolo[2,3d]pyrimidine.

[0453]4-(3-pyridylmethyl)amino-5,6-dimethyl-2-phenyl-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyrimidine.

[0454]4-(2-methylpropyl)amino-5,6-dimethyl-2-phenyl-7H-7-(1-phenylethyl)pyrrolo[2,3d]pyrimidine.

EXAMPLE 2

[0455] To a stirred suspension of triphenylphosphine (0.047 g, 0.179mmol) and benzoic acid (0.022 g, 0.179 mmol) in THF (1.0 mL) cooled to0° C. was added4-(4-trans-hydroxycyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine(0.05 g, 0.149 mmol) at 0° C. Diethyl azodicarboxylate (0.028 ml, 0.179mmol) was then added dropwise over 10 minutes. The reaction was thenallowed to warm to room temperature. After reaction was complete by TLCthe reaction mixture was quenched with aqueous sodium bicarbonate (3.0mL). The aqueous phase was separated and extracted with ether (2×5.0mL). The organic extracts were combined, dried, and concentrated invacuo to dryness. To the residue was added ether (2.0 mL) and hexane(5.0 mL) whereupon the bulk of the triphenylphosphine oxide was filteredoff. Concentration of the filtrate gave a viscous oil which was purifiedby column chromatography (hexane:ethyl acetate=4:1) to give 5.0 mg(7.6%) of4-(4-cis-benzoyloxycyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.MS (ES): 441.3 (M⁺+1). The reaction also produced 50.0 mg (84%) of4-(3-cyclohexenyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.MS (ES): 319.2 (M⁺+1).

EXAMPLE 3

[0456] To a solution of4-(4-cis-benzoyloxycyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine(5.0 mg, 0.0114 mmol) in ethanol (1.0 mL) was added 10 drops of 2Msodium hydroxide. After 1 hr, the reaction mixture was extracted withethyl acetate (3×5.0 mL) and the organic layer was dried, filtered andconcentrated in vacuo to dryness. The residue was subjected to columnchromatography (hexane:ethyl acetate=4:1) to give 3.6 mg (94%) of4-(4-cis-hydroxycyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.MS (ES): 337.2 (M⁺+1).

[0457] The following compounds were obtained in a similar manner as thatof Example 3:

[0458]4-(3-N,N-dimethyl-3-oxopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, CDCl₃) δ 2.01 (s, 3H), 2.31 (s, 3H), 2.73 (t, 2H), 2.97(s, 6H), 4.08 (m, 2H), 6.09 (t, 1H), 7.41-7.48 (m, 3H), 8.43 (m, 2H),10.46 (s, 1H); MS (ES): 338.2 (M⁺+1).

[0459]4-(2-formylaminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, CDCl₃) δ 2.26 (s, 3H), 2.37 (s, 3H), 3.59-3.78 (m, 2H),3.88-4.01 (m, 2H) 5.48-5.60 (m, 1H), 7.38-7.57 (m, 3H), 8.09 (s, 1H),8.30-8.45 (m, 2H), 8.82 (s, 1H); MS (ES) 310.1 (M⁺+1).

[0460]4-(3-acetylaminopropyl)amino-5,6-dimethyl-2-phenyl-⁷H-pyrrolo[2,3d]pyrimidine.MS (ES): 338.2 (M⁺+1).

EXAMPLE 4

[0461]4-(3-tert-butyloxy-3-oxopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine(70.0 mg, 0.191 mmol)) was dissolved in trifluoroaceticacid:dichloromethane (1:1, 5.0 mL). The resulting solution was stirredat room temperature for 1 hr. and then refluxed for 2 hr. After coolingdown to room temperature, the mixture was concentrated in vacuo todryness. The residue was subjected to preparative thin layerchromatography (EtOAc:hexane: AcOH=7:2.5:0.5) to give 40.0 mg (68%) of.4-(3-hydroxy-3-oxopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, CD₃OD) δ 2.32 (s, 3H), 2.38 (s, 3H), 2.81 (t, 2H), 4.01(t, 2H), 7.55 (m, 3H), 8.24 (m, 2H); MS (ES): 311.1 (M⁺+1).

[0462] The following compound was obtained in a similar manner as thatof Example 4:

[0463]4-(3-aminopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.MS (ES): 296.1 (M⁺+1), 279.1 (M⁺−NH₃).

EXAMPLE 5

[0464]4-(3-hydroxy-3-oxopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine(50.0 mg, 0.161 mmol) was dissolved in a mixture ofN,N-dimethylformamide (0.50 mL), dioxane (0.50 mL) and water(0.25 mL).To this solution was added methylamine (0.02 mL, 40% wt in water, 0.242mmol), triethylamine (0.085 mL) and N,N,N′N′-tetramethyl uroniumtetrafluoroborate (61.2 mg, 0.203 mmol). After stirring at roomtemperature for 10 min, the solution was concentrated and the residuewas subjected to preparative thin layer chromatography (EtOAc) to give35.0 mg (67%) of4-(3-N-methyl-3-oxopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, CDCl3) δ 1.92 (s, 3H), 2.30 (s, 3H), 2.65 (t, 2H), 4.08(t, 2H), 5.90 (t, 1H), 6.12 (m, 1H), 7.45 (m, 3H), 8.41 (m, 2H), 10.68(s, 1H); MS (ES): 311.1 (M⁺+1).

[0465] The following compounds were obtained in a similar manner as thatof Example 5:

[0466]4-(2-cyclopropanecarbonylaminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.MS (ES): 350.2 (M⁺+1).

[0467]4-(2-isobutyrylaminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.MS (ES): 352.2 (M⁺+1).

[0468]4-(3-propionylaminopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, CDCl₃) δ 1.00-1.08 (t, 3H), 1.71-2.03 (m, 4H), 2.08 (s,3H), 2.37 (s, 3H), 3.26-3.40 (m, 2H), 3.79-3.96 (m, 2H), 5.53-5.62 (m,1H), _(—)6.17-6.33 (m, 1H), 7.33-7.57 (m, 3H), 8.31-8.39 (m, 2H), 9.69(s, 1H); MS (ES): 352.2 (M⁺+1).

[0469]4-(2-methylsulfonylaminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, CDCl₃) δ 2.18 (s, 3H), 2.27 (s, 3H), 2.92 (s, 3H),3.39-3.53 (m, 2H), 3.71-3.88 (m, 2H), 5.31-5.39 (m, 1H), 6.17-6.33 (m,1H), 7.36-7.43 (m, 3H), 8.20-8.25 (m, 2H), 9.52 (s, 1H); MS (ES): 360.2(M⁺+1).

EXAMPLE 6

[0470] A mixture of 4-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d]pyrimidine (0.70 g, 2.72 mmol) and 1,2-diaminoethane (10.0 mL, 150 mmol)was refluxed under inert atmosphere for 6 hr. The excess amine wasremoved in vacuo, the residue was washed sequentially with ether andhexane to give 0.75 g (98%) of4-(2-aminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.MS (ES); 282.2 (M⁺+1), 265.1 (M⁺−NH₃).

EXAMPLE 7

[0471] To a solution of4-(2-aminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine(70.0 mg, 0.249 mmol) and triethylamine (50.4 mg, 0.498 mmol) indichloromethane (2.0 mL) was added propionyl chloride (25.6 mg, 0.024mL, 0.274 mmol) at 0° C. After 1 hr, the mixture was concentrated invacuo and the residue was subjected to preparative thin layerchromatography (EtOAc) to give 22.0 mg (26%) of4-(2-propionylaminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.MS (ES): 338.2 (M⁺+1).

[0472] The following compounds were obtained in a similar manner as thatof Example 7:

[0473]4-(2-N′-methylureaethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, CDCl₃) δ 2.13 (s, 3H), 2.32 (s, 3H), 3.53 (d, 3H), 3.55(m, 2H), 3.88 (m, 2H), 4.29 (m, 1H), 5.68 (t, 1H), 5.84 (m, 1H), 7.42(m, 3H), 8.36 (dd, 2H), 9.52 (s, 1H); MS (ES): 339.3 (M⁺+1).

[0474]4-(2-N′-ethylureaethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.MS (ES): 353.2 (M⁺+1).

EXAMPLE 8

[0475] To a solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (41.1 mg, 0.215 mmol), dimethylamino-pyridine (2.4 mg,0.020 mmol) and pyruvic acid (18.9 mg, 0.015 mL, 0.215 mmol) indichloromethane (2.0 mL) was added4-(2-aminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d] pyrimidine(55.0 mg, 0.196 mmol). The mixture was stirred at room temperature for 4hr. Usual workup and column chromatography (EtOAc) then gave 10.0 mg(15%) of 4-(2′-pyruvylamidoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine. MS (ES): 352.2 (M⁺+1).

EXAMPLE 9

[0476] To a solution of4-(2-aminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine(60.0 mg, 0.213 mmol) in dichloromethane (2.0 mL) was addedN-trimethylsilyl isocyanate (43.3 mg, 0.051 mL, 0.320 mmol). The mixturewas stirred at room temperature for 3 hr followed by addition of aqueoussodium bicarbonate. After filtration through small amount of silica gel,the filtrate was concentrated in vacuo to dryness to give 9.8 mg (14%)of4-(2-ureaethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.MS (ES): 325.2 (M⁺+1).

[0477] The following compounds were obtained in a similar manner as thatof Example 9:

[0478]dl-4-(2-acetylaminopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, CDCl₃) δ 1.28-1.32 (d, J=8 Hz, 3H), 1.66 (s, 3H), 1.96(s, 3H), 2.30 (s, 3H) 3.76-3.83 (m, 2H), 4.10-4.30 (m, 1H), 5.60-5.66(t, J=6 Hz, 1H), 7.40-7.51(m, 3H), 8.36-8.43 (m, 2H), 10.83 (s, 1H); MS(ES): 338.2 (M⁺+1).

[0479](R)-4-(2-acetylaminopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, CDCl₃) δ 1.31 (d, 3H), 1.66 (s, 3H) 1.99 (s, 3H), 2.31(s, 3H), 3.78-3.83 (m, 2H), 4.17-4.22 (m, 1H), 5.67 (t, 1H), 7.38-7.5(m, 3H), 8.39 (m, 2H), 10.81 (s, 1H); MS (ES): 338.2 (M⁺+1).

[0480](R)-4-(1-methyl-2-acetylaminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, CDCl₃) δ 1.41 (d, 3H), 1.68 (s, 3H), 2.21 (s, 3H), 2.34(s, 3H), 3.46-3.52 (br, m, 2H), 4.73 (m, 1H), 5.22 (d, 1H), 7.41-7.46(m, 3H), 8.36-8.40 (m, 2H), 8.93 (s, 1H); MS (ES): 338.2 (M⁺+1).

[0481](S)-4-(2-acetylaminopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, CDCl₃) δ 1.31 (d, 3H), 1.66 (s, 3H) 2.26 (s, 3H), 2.35(s, 3H), 3.78-3.83 (m, 2H), 4.17-4.22 (m, 1H), 5.67 (t, 1H), 7.38-7.5(m, 3H), 8.39 (m, 2H), 8.67(s, 1H); MS (ES): 338.2 (M⁺+1).

[0482](S)-4-(1-methyl-2-acetylaminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, CDCl) δ 1.41 (d, 3H), 1.68 (s, 3H), 2.05 (s, 3H), 2.32(s, 3H), 3.46-3.52 (m, 2H), 4.73 (m, 1H), 5.22 (d, 1H), 7.41-7.46 (m,3H) 8.36-8.40 (m, 2H), 10.13 (s, 1H); MS (ES): 338.2 (M⁺+1).

EXAMPLE 10

[0483] Reaction of4-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine with themixture of dl-1-amino-2-(1,1-dimethyl ethoxy)carbonylamino-propane anddl-2-amino-1-(1,1-dimethyl ethoxy)carbonylamino-propane was run in asimilar manner as that of Example 1. The reaction gave a mixture ofdl-4-(1-methyl-2-(1,1-dimethylethoxy)carbonylamino)ethylamino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidineanddl-4-(2-methyl-2-(1,1-dimethylethoxy)carbonylamino)ethylamino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidinewhich were separated by column chromatography (EtOAc:hexanes=1:3). Thefirst fraction was dl-4-(1-methyl-2-(1,1-dimethylethoxy)carbonylaminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine:¹H NMR (200 MHz, CDCl,) δ 1.29-1.38 (m, 12H), 1.95 (s, 3H), 2.31 (s, 3H)3.34-3.43 (m, 2H), 4.62-4.70 (m, 1H), 5.36-5.40 (d, J=8 Hz, 1H), 5.53(br, 1H), 7.37-7.49(m, 3H), 8.37-8.44(m, 2H), 10.75 (s, 1H). MS 396.3(M⁺+1); The second fraction was dl-4-(2-(1,1-dimethylethoxy)carbonylaminopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine:¹H NMR (200 MHz, CDCl₃) δ 1.26-1.40 (m, 12H), 2.00 (s, 3H), 2.31 (s, 3H)3.60-3.90 (m, 2H), 3.95-4.10 (m, 1H), 5.41-5.44 (d, J=6.0 Hz, 1H),5.65(br, 1H), 7.40-7.46(m, 3H), 8.37-8.44(m, 2H), 10.89 (s, 1H); MS(ES): 396.2 (M⁺+1).

[0484] The following compounds were obtained in a similar manner as thatof Example 10:

[0485](S,S)-4-(2-acetylaminocyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, CDCl₃) δ_(—)1.43 (m, 4H), 1.60 (s, 3H), 1.83 (m, 2H),2.18 (s, 3H), 2.30 (m, 2H), 2.32 (s, 3H), 3.73 (br, 1H), 4.25 (br, 1H),5.29 (d, 1H), 7.43-7.48 (m, 3H), 8.35-8.40 (m, 2H), 9.05 (s, 1 H).

[0486]4-(2-methyl-2-acetylaminopropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, CDCl₃) δ 1.51 (s, 6H), 1.56 (s, 3H), 2.07 (s, 3H), 2.36(s, 3H), 3.76 (d, 2H), 5.78 (t, 1H), 7.41-7.48 (m, 3H), 7.93 (s, 1H),8.39 (m, 2H), 10.07 (s, 1H); MS (ES): 352.3 (M⁺+1).

EXAMPLE 11

[0487] dl-4-(1-methyl-2-(1,1-dimethylethoxy) carbonyl aminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine (60.6 mg, 0.153mmol) was treated with trifluoroacetic acid (0.5 mL) in dichloromethane(2.0 mL) for 14 hr. The organic solvent was removed in vacuo to dryness.The residue was dissolved in N,N-dimethylformamide (2.0 mL) andtriethylamine (2.0 mL). To the solution at 0° C. was added aceticanhydride (17.2 mg, 0.016, 0.169 mmol). The resulted mixture was stirredat room temperature for 48 hr and then concentrated in vacuo to dryness.The residue was subjected to preparative thin layer chromatography(EtOAc) to give 27.0 mg (52%) ofdl-4-(1-methyl-2-acetylaminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, CDCl₃) δ 1.38-1.42 (d, J=8 Hz, 3H), 1.69 (s, 3H), 2.01(s, 3H), 2.32 (s, 3H) 3.38-3.60 (m, 2H), 4.65-4.80 (m, 1H), 5.23-5.26(d, J=6 Hz, 1H), 7.40-7.51(m, 3H), 8.37-8.43(m, 2H), 10.44 (s, 1H); MS(ES): 338.2 (M⁺+1).

EXAMPLE 12

[0488](R,R)-4-(2-aminocyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine,prepared in a similar manner as that of Example 1 from4-chloro-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine (0.15 g, 0.583mmol) and (1R, 2R)-(-)-1,2-diaminocyclohexane (0.63 g, 5.517 mmol), wastreated with triethylamine (0.726 g, 7.175 mmol) and acetic anhydride(0.325 g, 3.18 mmol) in N,N-dimethylformamide (10.0 mL) at roomtemperature for 2 hr. After removal of solvent in vacuo, ethyl acetate(10.0 mL) and water (10.0 mL) were added to the residue. The mixture wasseparated and the aqueous layer was extracted with ethyl acetate (2×10.0mL). The combined ethyl acetate solution was dried (MgSO₄) and filtered.The filtrate was concentrated in vacuo to dryness and the residue wassubjected to column chromatography (EtOAc:Hexane=1:1) to give 57.0 mg(26%) of(R,R)-4-(2-acetylaminocyclohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.¹H NMR (200 MHz, CDCl₃) δ_(—)1.43 (m, 4H), 1.60 (s, 3H), 1.84 (m, 2 H),2.22 (s, 3H), 2.30 (m, 2H), 2.33 (s, 3H), 3.72 (br, 1H), 4.24 (br, 1H),5.29 (d, 1H), 7.43-7.48 (m, 3H), 8.35-8.39 (m, 2H), 8.83 (s, 1H); MS(ES): 378.3 (M⁺+1).

EXAMPLE 13

[0489] To a solution of4-(2-hydroxyethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine(40.0 mg, 0.141 mmol) in pyridine (1.0 mL) was added acetic anhydride(0.108 g, 1.06 mmol) at 0° C. The mixture was stirred at roomtemperature for 4 hr and the solvent was removed in vacuo. The residuewas subjected to preparative thin layer chromatography(EtOAc:hexane=1:1) to give 32.3 mg (71) of 4-(2-acetyloxyethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo [2,3d] pyrimidine. ¹H NMR (200MHz, CDCl₃) δ 1.90 (s, 3H), 2.08 (s, 3H), 2.31 (s, 3H), 4.05 (m, 2H),4.45 (t, 2H), 5.42 (m, 1H), 7.41-7.49 (m, 3H), 8.42(m, 2H), 11.23 (s,1H).

EXAMPLE 14

[0490] A solution of Fmoc-β-Ala-OH (97.4 mg, 0.313 mmol) and oxalylchloride (39.7 mg, 27.3 μL, 0.313 mmol) in dichloromethane (4.0 mL) with1 drop of N,N-dimethylformamide was stirred at 0° C. for 1 hr followedby addition of4-(2-aminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine(80.0 mg, 0.285 mmol) and triethylamine (57.6 mg, 79.4 μL, 0.570 mmol)at 0° C. After 3 hr, the mixture was concentrated in vacuo and theresidue was treated with the solution of 20% piperidine inN,N-dimethylforamide (2.0 mL) for 0.5 hr. After removal of the solventin vacuo, the residue was washed with diethyl ether:hexane (1:5) to give3.0 mg (3%) of4-(6-amino-3-aza-4-oxohexyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.MS (ES): 353.2 (M⁺+1).

EXAMPLE 15

[0491] A solution of4-(2-aminoethyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine(70.0 mg, 0.249 mmol) and succinic anhydride (27.0 mg, 0.274 mmol) indichloromethane (4.0 mL) with 1 drop of N,N-dimethylformamide wasstirred at room temperature for 4 hr. The reaction mixture was extractedwith 20% sodium hydroxide (3×5.0 mL). The aqueous solution was acidifiedwith 3 M hydrochloride to pH=7.0. The whole mixture was extracted withethyl acetate (3×10 mL). The combined organic solution was dried (MgSO₄)and filtered. The filtrate was concentrated in vacuo to dryness to give15.0 mg (16%) of4-(7-hydroxy-3-aza-4,7-dioxoheptyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.MS (ES): 382.2 (M⁺+1).

EXAMPLE 16

[0492] To 10 mL of dimethylformamide (DMF) at room temperature wereadded 700 mg of4-cis-3-hydroxycyclopentyl)amino-2-phenyl-5,6-dimethyl-7H-pyrrolo[2,3d]pyrimidinefollowed by 455 mg of N-Boc glycine, 20 mg of N,N-dimethylaminopyridine(DMAP), 293 mg of hydroxybenzotriazole (HOBT) and 622 mg of1-(3-dimethylaminopropyl)-3-ethylcarboiimide hydrochloride (EDCl). Thereaction mixture was left stirring overnight. DMF was then removed underreduced pressure and the reaction mixture was partitioned between 20 mLof ethyl acetate and 50 mL of water. The aqueous portion was extractedfurther with 2×20 mL of ethyl acetate and the combined organic portionswere washed with brine, dried over anhydrous sodium sulfate, filteredand concentrated. Purification on silica gel, eluting with ethylacetate/hexane gave 410 mg of the desired product:4-(cis-3-(N-t-butoxycarbonyl-2-aminoacetoxy) cyclopentyl)amino-2-phenyl-5,6,-dimethyl-7H-pyrrolo[2,3d] pyrimidine, MS (ES)(M⁺+1)=480.2. The ester was then treated with 5 mL of 20%trifluoroacetic acid in dichloromethane at room temperature, left overnight and then concentrated. Trituration with ethyl acetate gave 300 mgof an off white solid;4-(cis-3-(2-aminoacetoxy)cyclopentyl)amino-5,6-dimethy1-2-phenyl-7H-pyrrolo[2,3d]pyrimidine trifluoroacetic acid salt, MS (ES)(M⁺+1)=380.1.

[0493] One skilled in the art will appreciate that the followingcompounds can be synthesized by the methods disclosed above:

[0494] 4-(cis-3-hydroxycyclopentyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d] pyrimidine MS (ES)(M⁺+1)=323.1.

[0495]4-(cis-3-(2-aminoacetoxy)cyclopentyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidinetrifluoroacetic acid salt MS (ES) (M⁺+1)=380.1.

[0496]4-(3-acetamido)piperidinyl-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidineMS (ES) (M⁺+1)=364.2.

[0497]4-(2-N′-methylureapropyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine, MS (ES) (M⁺+1)=353.4.

[0498]4-(2-acetamidobutyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine,MS (ES) (M⁺+1)=352.4.

[0499]4-(2-N′-methylureabutyl)amino-5,6-dimethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine,MS (ES) (M⁻+1)=367.5.

[0500]4-(2-aminocyclopropylacetamidoethyl)amino-2-phenyl-7H-pyrrolo[2,3d]pyrimidine MS (ES) (M⁻+1)=309.1.

[0501]4-(trans-4-hydroxycyclohexyl)amino-2-(3-chlorophenyl)-7H-pyrrolo[2,3d]pyrimidine MS (ES) (M+1)=342.8.

[0502]4-(trans-4-hydroxycyclohexyl)amino-2-(3-fluorophenyl)-7H-pyrrolo[2,3d]pyrimidine MS (ES) (M⁺+1)=327.2.

[0503]4-(trans-4-hydroxycyclohexyl)amino-2-(4-pyridyl)-7H-pyrrolo[2,3d]pyrimidineMS (ES) (M⁺+1)=310.2.

EXAMPLE 17

[0504]

[0505] The pyrrole nitrogen of (7) (Scheme IX) was protected withdi-t-butyldicarbonate under basic conditions to yield the correspondingcarbamate (22). Radical bromination of (22) proceeded regioselectivelyto yield bromide (23). In general, compound (23) served as a keyelectrophilic intermediate for various nucleophilic coupling partners.

[0506] Displacement of the alkyl bromide with sodium phenolatetrihydrate yielded compound (24). Subsequent displacement of the arylchloride and removal of the t-butyl carbamate protecting group occurredin one step yielding desired compound (25).

[0507] Detailed Synthesis of Compounds (22)-(25) in Accordance withScheme IX

[0508] Di-t-butyl dicarbonate (5.37 g, 24.6 mmol) and dimethylaminopyridine (1.13 g, 9.2 mmol) were added to a solution containing (7)(1.50 g, 6.15 mmol) and pyridine (30 mL) After 20 h the reaction wasconcentrated and the residue was partitioned between CH₂Cl₂ and water.The CH₂Cl₂ layer was separated, dried over MgSO₄, filtered andconcentrated to yield a black solid. Flash chromatography (SiO₂; 1/9EtOAc/Hexanes, R_(f) 0.40) yielded 1.70 g (80%) of a white solid (22).¹H NMR (200 MHz, CDCl₃) δ 8.50 (m, 2H, Ar—H), 7.45 (m, 3H, Ar—H), 6.39(s, 1H, pyrrole-H), 2.66 (s, 3H,pyrrole-CH₃), 1.76 (s, 9H,carbamate-CH₃); MS, M+1=344.1; Mpt=175-177° C.

[0509] N-Bromosuccinimide (508 mg, 2.86 mmol) and AIBN (112 mg, 0.68mmol) were added to a solution containing (22) (935 mg, 2.71 mmol) andCCl₄ (50 mL). The solution was heated to reflux. After 2 h the reactionwas cooled to room temperature and concentrated in vacuo to yield awhite solid. Flash chromatography (SiO₂; 1/1 CH₂Cl₂/Hexanes, R_(f) 0.30)yielded 960 mg (84%)of a white solid (23). ¹H NMR (200 MHz, CDCl₃)δ_(—)8.52 (m, 2H, Ar—H), 7.48 (m, 3H, Ar—H), 6.76 (s, 1H, pyrrole-H),4.93 (s, 2H, pyrrole-CH₂Br), 1.79 (s, 9H, carbamate-CH₃); MS, M+1=423.9;Mpt=155-157° C.

[0510] Sodium phenoxide trihydrate (173 mg, 1.02 mmol) was added in oneportion to a solution of bromide (23) (410 mg, 0.97 mmol) dissolved inCH₂Cl₂ (5 mL) and DMF (10 mL). After 2 h the reaction solution waspartitioned between CH₂Cl₂ and water. The water layer was extracted withCH₂Cl₂. The combined CH₂Cl₂ layers were washed with water, dried overMgSO₄, filtered and concentrated to yield a yellow solid. Flashchromatography (SiO₂; 1/6 EtOAc/Hexanes, R_(f) 0.30) yielded 210 mg(50%) of a white solid (24). ¹H NMR (200 MHz, CDCl₃) δ_(—)8.53 (m, 2H,Ar—H), 7.48 (m, 3H, Ar—H), 7.34 (m, 2H, Ar—H), 7.03 (m, 3H, Ar—H), 6.83(s, 1H, pyrrole-H), 5.45 (s, 2H, ArCH₂O), 1.76 (s, 9H, carbamate-CH₃);MS, M =436.2.

[0511] A solution containing (24) (85 mg, 0.20 mmol),N-acetylethylenediamine (201 mg, 1.95 mmol) and DMSO (3 mL) was heatedto 100° C. After 1 h the temperature was raised to 130° C. After 3 h thereaction was cooled to room temperature and partitioned between EtOAcand water. The water layer was extracted with EtOAc (2×). The combinedEtOAc layers are washed with water, dried over MgSO₄, filtered andconcentrated. Flash chromatography (SiO₂; 1/10 EtOH/ CHCl₃₁ R_(f) 0.25)yielded 73 mg (93%)of a white foamy solid (25). ¹H NMR (200 MHz,DMSO-d₆) δ 11.81 (br s, 1H, N-H), 8.39 (m, 2H, Ar—H), 8.03 (br t, 1H1,N-H), 7.57 (br t, 1H, N—H), 7.20-7 50 (m, 5H, Ar—H), 6.89-7.09 (m, 3 H,Ar—H), 6.59 (s, 1H, pyrrole-H), 5.12 (s, 2H, ArCH₂O), 3.61 (m, 2H,NCH₂), 3.36 (m, 2H, NCH₂), 1.79 (s, 3H, COCH₃); MS, M+1=402.6.

[0512] The following compounds were obtained in a manner similar to thatof Example 17:

[0513]4-(2-acetylaminoethyl)amino-6-phenoxymethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.mp 196-197 C; MS (ES): 401.6 (M⁺+1).

[0514]4-(2-acetylaminoethyl)amino-6-(4-fluorophenoxy)methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.MS(ES): 420.1 (M⁻+1).

[0515]4-(2-acetylaminoethyl)amino-6-(4-chlorophenoxy)methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.MS(ES): 436.1 (M⁻+1)

[0516]4-(2-acetylaminoethyl)amino-6-(4-methoxyphenoxy)methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.MS(ES): 432.1 (M⁻+1).

[0517]4-(2-acetylaminoethyl)amino-6-(N-pyridin-2-one)methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.MS(ES): 403.1 (M⁺+1).

[0518]4-(2-acetylaminoethyl)amino-6-(N-phenylamino)methyl-2-phenyl-7H-pyrrolo[2,3]pyrimidine.MS (ES): 400.9 (M⁺+1).

[0519]4-(2-acetylaminoethyl)amino-6-(N-methyl-N-phenylamino)methyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.MS(ES): 414.8 (M⁺+1).

[0520]4-(2-N′-methylureaethyl)amino-6-phenoxymethyl-2-phenyl-7H-pyrrolo[2,3d]pyrimidine.MS (ES): 416.9 (M⁺+1).

EXAMPLE 18

[0521] Synthesis of Adenosine A_(2a) Antagonists, compounds 1601, 1602,and 1603.

[0522] Compound 26 (10.93g, 50.76 mmol) was dissolved in DMF (67 mL).4-Amidinopyridine hydrochloride (8.0g, 50.76 mmol) and DBU (15.4 g,101.5 mmol) were added sequentially and the reaction was heated to 85°C. After 22 hours, the reaction was cooled to room temperature and theDMF was removed in vacuo. The dark oil was diluted with 2M HCl (80 mL).The reaction was allowed to stand. After 2 hours, the solution wascooled to 10° C. and filtered. The solid was washed with cold water anddried to yield 7.40g of a yellow solid, compound 27 (69%). ¹H-NMR (200MHz, d₆-DMSO) d 6.58 (s, 1H), 7.27 (s, 1H), 8.53 (d, 2H, J=5.6), 9.00(d, 2H, J=5.2 Hz), 12.35 (brs, 1H). MS (ES): 212.8 (M⁺+1).

[0523] Compound 27 (7.4 mmol, 29.8 mmol) was diluted with POCl₃ andheated to 105° C. After 18 hours, the reaction is cooled to roomtemperature and the POCl₃ is removed in vacuo. The thick dark oil isdiluted with MeOH (75 mL) followed by ether (120 mL). The amorphous redsolid is filtered and washed with ether to yield 3.82 g of a red solid.The crude solid, compound 28, is approximately 80% pure and used withoutfurther purification in the next reaction. ¹H-NMR (200 MHz, d₆-DMSO) d6.58 (s, 1H), 7.27 (s, 1H), 8.53 (d, 2H, J=5.6), 9.00 (d, 2H, J=5.2 Hz),12.35 (brs, 1H). MS (ES): 212.8 (M⁺+1).

[0524] Compound 1601: DMSO (5 mL) and D-prolinol (500 mg, 4.94 mmol)were added to compound 28 (500 mg, 2.17 mmol) was added. The reactionwas heated to 120° C. After 18 hours, The reaction was cooled to roomtemperature and diluted with EtOAc and H₂O. The layers were separatedand the aqueous layer was extracted with EtOAc (2×). The combinedorganic layers were washed with H₂O (2×), brine, dried over MgSO₄,filtered and concentrated to yield 200 mg of a tan solid. The solid wasrecrystallized from EtOAc to yield 82 mg of a tan solid (13%). ¹H-NMR(200 MHz, d₆-DMSO) d 2.05 (m, 4H), 3.43 (m, 1H), 3.70-4.00 (m, 3H), 4.50(brs, 1H), 4.92 (brs, 1H), 6.62 (m, 1H), 7.22 (m, 1H), 8.22 (d, 2H,J=6.0 Hz), 6.64 (d, 2H, J=6.2 Hz), MS (ES): 296.0 (M⁺+1), mp =210-220°C. (decomp.).

[0525] Compound 1602: Chromatography (silica, 9:1 CHCl3/MeOH) yielded 10mg of a tan solid (2%).¹H-NMR (d₆-DMSO) d 2.00-2.50 (m, 4H), 4.05 (m,1H), 4.21 (m, 1H), 6.71 (d, 1H, J=3.2 Hz), 7.18 (d, 1H, J=3.2 Hz), 8.37(d, 2H, J=4.8 Hz), 8.56 (d, 2H, J=5.0 Hz). MS (ES): 309.1 (M⁻+1).

[0526] Compound 1603. Chromatography (silica, 20:1 Hexanes /EtOAc)yielded 135 mg of a tan solid (53%). H-NMR (dE-DMSO) d 2.00 (m, 4H),3.43 (brs, 1H), 3.74 (brs, 2H), 3.87 (brs, 1H), 4.49 (brs, 1H), 4.93 (m,1H), 6.56 (m, 1H), 7.12 (m, 1H), 7.40 (m, 3H), 8.34 (m, 2H), 11.62 (brs,1H). MS (ES): 295.1 (M⁻+1).

[0527] Compound 1605. Into a 50 mL REF 60 mg of2-(4′-pyridyl)-4-Chloropyrimidinopyrrole HCl salt was dissolved in 2 mLanhydrous DMSO. 3-(R)-Hydroy-(D)-prolinol TFA salt (380 mg) and 500 mgsodium bicarbonate were added thereto. The mixture was then flashed withnitrogen gas for 5 min and heated to 130° C. After 2 hours, the reactionwas cooled to room temperature and the DMSO was removed in vacuo. Theresidue was partitioned between EtOAc (15 mL) and saturated sodiumbicarbonate aqueous solution (15 mL). The organic layer was separatedand washed with brine (15 mL) and dried over NaSo,. After removal ofsolvent, the crude product was purified by preparative TLC(CH₂Cl₂/MeOH=95/5) to yield 35 mg (50%). ¹H-NMR (200 MHz, CDCl) (2.3-2.5(1H), 3.4-3.8 (3H), 4.4-4.6 (2H), 6.4 (1H); 7.1 (1H); 8.2 (d, 2H); 8.7(d, 2H); 11.0 (1H). MS (ES): 312 (M⁺+1).

EXAMPLE 19

[0528] Synthesis of Adenosine A_(2a) Antagonist, compound 1606.

[0529] Compound 28 (200 mg) was treated with DMF (30 mL), (,(-dimethylglycine methyl ester (73 mg HCl salt in 2 mL water) and 500 mgsodium bicarbonate. After 18 hours, the DMF was removed in vacuo. Theresidue was partitioned between EtOAc (30 mL) and saturated sodiumbicarbonate aqueous solution (15 mL). The organic layer was washed withbrine (15 mL), dried over sodium sulfate, filtered and concentrated.Chromatography (silica, 10:4 hexanes/EtOAc) yielded 150 mg of pureproduct, compound 29 (69%). ¹H-NMR (200 MHz, CDCl₃), ( 1.4 (s, 6H), 3.8(s, 3H); 3.9 (s, 2H); 6.4 (s, 1H); 7.4-7.5 (m, 3H); 8.4 (m, 2H); 9.8 (s,1H)

[0530] Compound 1606:

[0531] Procedure is the same as Compound 1605 (72%). ¹H-NMR (200 MHz,CDCl₃), (1.3 (s, 6H), 1.7-1.9 (m, 2H); 2.05-2.30 (m, 2H); 3.6-4.1 (m,11H); 4.80-4.95 (m, 1H); 6.4 (s, 1H); 7.4-7.6 (m, 3H); 8.3-8.4 (d, J=8.5Hz, 2H), 10 (s, 1H). MS (ES): 424.0 (M⁺+1).

[0532] The following compounds can be synthesized in the same manner.

[0533] Compound 1600: (51%). MS (ES): 326.0 (M⁺+1).

[0534] Compound 1607: ¹H-NMR (200 MHz, CDCl₃), (1.40-1.80 (m, 5H)2.80-3.50 (m, 3H), 4.60-4.80 (m, 3H), 6.66 (d, 1H, J=6.2 Hz), 7.26 (m,1H), 8.21 (d, 2H, J=6.3 Hz), 8.65 (d, 2H, J=5.8 Hz), 11.90 (s, 1H). MS(ES): 310.1 (M⁺+1).

[0535] Compound 1608: (64%). ¹H-NMR (200 MHz, d₆-DMSO), (1.75 (s, 3H),2.11 (s, 3H), 2.29 (s, 3H), 3.56 (m, 6H), 7.23-7.41 (m, 5H), 8.00 (brs,1H), 8.23 (d, 2H, J=6.0 Hz), 8.63 (d, 2H, J=5.4 Hz), 8.82 (brs, 1H),11.56 (brs, 1H). MS (ES): 444.0 (M⁺+1).

[0536] Compound 1604: ¹H-NMR (200 MHz, CD₃OD) (3.40 (m, 4H), 4.29 (m,4H), 6.99 (s, 1H), 7.5-7.2 (m, 3H), 7.90 (d, 2H), 8.39 (d, 2H), 8.61 (d,2H). MS (ES): 357.0 (M⁺+1).

[0537] Yeast β-Galactosidase Reporter Gene Assays for Human Denosine A₁and A_(2a) Receptor:

[0538] Yeast strains (S. cerevisiae) were transformed with humanadenosine A₁ (A₁R; CADUS strain CY12660) or human A_(2a) (A_(2a); CADUSstrain CY8362) and the addition of a lacZ(β-Galactosidase) reporter geneto utilize as a functional readout. A complete description of thetransformations is listed below (see Yeast Strains). NECA(5′-N-ethylcarboxamidoadenosine), a potent adenosine receptor agonistwith similar affinity for A₁ and A_(2a) receptors, was used as a ligandfor all assays. Test compounds were examined at 8 concentrations(0.1-10,000 nM) for ability to inhibit NECA-induced β-Galactosidaseactivity by CY12660 or CY8362.

[0539] Preparation of Yeast Stock Cultures:

[0540] Each of the respective yeast strains, CY12660 and CY8362, werestreaked onto an LT agar plate and incubated at 30° C. until colonieswere observed. Yeast from these colonies were added to LT liquid (pH6.8) and grown overnight at 30° C. Each yeast strain was then diluted toan OD₆₀₀=1.0-2.0 (approximately 1-2×10⁷ cells/ml), as determinedspectrophotometrically (Molecular Devices VMAX). For each 6 ml of yeastliquid culture, 4 ml of 40% glycerol (1:1.5 vol:vol) was added(“yeast/glycerol stock”). From this yeast/glycerol stock, ten 1 mlaliquots were prepared and stored at −80° C. until required for assay.

[0541] Yeast A₁ R and A_(2a)R Assay:

[0542] One vial each of CY8362 and CY12660 yeast/glycerol stock wasthawed and used to inoculate Supplemented LT liquid media, pH 6.8 (92 mlLT liquid, to which is added: 5 ml of 40% glucose, 0.45 ml of 1M KOH and2.5 ml of Pipes, pH 6.8). Liquid cultures were grown 16-18 hr(overnight) at 30° C. Aliquots from overnight cultures were then dilutedin LT media, containing 4U/ml adenosine deaminase (Type VI or VII fromcalf intestinal mucosa, Sigma), to obtain OD₆₀₀=0.15 (1.5×10⁶ cells/ml)for CY8362 (A2aR) and OD₆₀₀=0.50 (5×10⁶ cells/ml) for CY12660 (A₁R).

[0543] Assays were conducted with a final volume of 100 ul in 96-wellmicrotiter plates, such that a final concentration of 2% DMSO wasachieved in all wells. For primary screening, 1-2 concentrations of testcompounds were utilized (10 uM, 1 μM ). For compound profiling, 8concentrations were tested (10000, 1000, 500, 100, 50, 10, 1 and 0.1nM). To each microtiter plate, 10 ul of 20% DMSO was added to “Control”and “Total” wells while 10 ul of Test Compound (in 20% DMSO) was addedto “Unknown” wells. Subsequently, 10 ul of NECA (5 uM for A₁R, 1 uM forA_(2a)R) were added to “Total” and “Unknown” wells; 10 ul of PBS wasadded to the “Control” wells. In the final addition, 80 ul of yeaststrain, CY8362 or CY12660, were added to all wells. All plates were thenagitated briefly (LabLine orbital shaker 2-3 min) and allowed toincubate for 4 hrs. at 30° C. in a dry oven.

[0544] β-Galactosidase activity can be quantitated using eithercalorimetric (e.g., ONPG, CPRG), luminescent (e.g., Galacton-Star) orfluorometric substrates (e.g., FDG, Resorufin) substrates. Currently,fluorescence detection is preferred on the basis of superiorsignal:noise ratio, relative freedom from interference and low cost.Fluorescein digalactopyranoside (FDG, Molecular Probes or Marker GeneTechnologies), a fluorescent β-Galactosidase substrate, was added to allwells at 20 ul/well (final concentration=80 uM). Plates were shaken for5-6 sec (LabLine orbital shaker) and then incubated at 37° C. for 90 min(95% O₂/5% CO₂ incubator). At the end of the 90 min incubation period,β-Galactosidase activity was stopped using 20 ul/well of 1M Na₂CO₃ andall plates shaken for 5-6 sec. Plates were then agitated for 6 sec andrelative fluorescence intensity determined using a fluorometer (TecanSpectrafluor; excitation=485 nm, emission=535 nm).

[0545] Calculations: Relative fluorescence values for “Control” wellswere interpreted as background and subtracted from “Total” and “Unknown”values. Compound profiles were analyzed via logarithmic transformation(x-axis: compound concentration) followed by one site competition curvefitting to calculate IC₅₀ values (GraphPad Prism)

[0546] Yeast strains: Saccharomyces cerevisiae strains CY12660[far1*1442 tbt1-1 fus1-HIS3 can1 ste14::trp1::LYS2 ste3*1156gpa1(41)-Gαi3 lys2 ura3 leu2 trp1: his3; LEU2PGKp-Mfα1Leader-hA1R-PHO5term 2mu-orig REP3 Ampr] and CY8362[gpa1p-rGαsE10K far1*1442 tbt1-1 fusl-HIS3 can1 ste14::trp1: LYS2ste3*1156 lys2 ura3 leu2 trp1 his3; LEU2 PGKp-hA2aR 2mu-ori REP3 Ampr]were developed.

[0547] LT Media: LT (Leu-Trp supplemented) media is composed of 100 gDIFCO yeast nitrogen base, supplemented with the following: 1.0 gvaline, 1.0 g aspartic acid, 0.75 g phenylalanine, 0.9 g lysine, 0.45 gtyrosine, 0.45 g isoleucine, 0.3 g methionine, 0.6 g adenine, 0.4 guracil, 0.3 g serine, 0.3 g proline, 0.3 g cysteine, 0.3 g arginine, 0.9g histidine and 1.0 g threonine.

[0548] Construction of Yeast Strains Expressing Human A₁ AdenosineReceptor

[0549] In this example, the construction of yeast strains expressing ahuman A₁ adenosine receptor functionally integrated into the yeastpheromone system pathway is described.

[0550] I. Expression Vector Construction

[0551] To construct a yeast expression vector for the human A₁ adenosinereceptor, the A₁ adenosine receptor cDNA was obtained by reversetranscriptase PCR of human hippocampus mRNA using primers designed basedon the published sequence of the human A₁ adenosine receptor andstandard techniques. The PCR product was subcloned into the NcoI andXbaI sites of the yeast expression plasmid pMP15.

[0552] The pMP15 plasmid was created from pLPXt as follows: The XbaIsite of YEP51 (Broach, J. R. et al. (1983) “Vectors for high-level,inducible expression of cloned genes in yeast” p. 83-117 in M. Inouye(ed.), Experimental Manipulation of Gene Expression. Academic Press, NewYork) was eliminated by digestion, end-fill and religation to createYep51NcoDXba. Another XbaI site was created at the BamHI site bydigestion with BamHI, end-fill, linker (New England Biolabs, # 1081)ligation, XbaI digestion and re-ligation to generate YEP51NcoXt. Thisplasmid was digested with Esp31 and NcoI and ligated to Leu2 and PGKpfragments generated by PCR. The 2 kb Leu2 PCR product was generated byamplification from YEP51Nco using primers containing Esp3l and BglIIsites. The 660 base pair PGKp PCR product was generated by amplificationfrom pPGKas (Kang, Y. -S. et al. (1990) Mol. Cell. Biol. 10:2582-2590)with PCR primers containing BglII and NcoI sites. The resulting plasmidis called pLPXt. pLPXt was modified by inserting the coding region ofthe a-factor prepro leader into the NcoI site. The prepro leader wasinserted so that the NcoI cloning site was maintained at the 3′ end ofthe leader, but not regenerated at the 5′ end. In this way receptors canbe cloned by digestion of the plasmid with NcoI and XbaI. The resultingplasmid is called pMP15.

[0553] The pMP15 plasmid into which was inserted the human A₁ adenosinereceptor cDNA was designated p5095. In this vector, the receptor cDNA isfused to the 3′ end of the yeast a-factor prepro leader. During proteinmaturation the prepro peptide sequences are cleaved to generate maturefull-length receptor. This occurs during processing of the receptorthrough the yeast secretory pathway. This plasmid is maintained by Leuselection (i.e., growth on medium lacking leucine). The sequence of thecloned coding region was determined and found to be equivalent to thatin the published literature (GenBank accession numbers S45235 andS56143).

[0554] II. Yeast Strain Construction

[0555] To create a yeast strain expressing the human A₁ adenosinereceptor, yeast strain CY7967 was used as the starting parental strain.The genotype of CY7967 is as follows:

[0556] MATA gpaD1163 gpa1(41)Gαi3 far1D1442 tbt-1 FUS1-HIS3 can1ste14::trp1::LYS2 ste3D1156 lys2 ura3 leu2 trp1 his3

[0557] The genetic markers are reviewed below:

[0558] MATa . . . Mating type a.

[0559] gpa1D1163 . . . The endogenous yeast G-protein GPA1 has beendeleted.

[0560] gpa1(41)Gαi3 . . . gpa1(41)-Gαi3 was integrated into the yeastgenome. This chimeric Ga protein is composed of the first 41 amino acidsof the endogenous yeast Ga subunit GPA1 fused to the mammalian G-proteinGαi3 in which the cognate N-terminal amino acids have been deleted.

[0561] far1D1442 . . . FAR1 gene (responsible for cell cycle arrest) hasbeen deleted (thereby preventing cell cycle arrest upon activation ofthe pheromone response pathway).

[0562] tbt-1 . . . strain with high transformation efficiency byelectroporation.

[0563] FUS1-HIS3 . . . a fusion between the FUS1 promoter and the HIS3coding region (thereby creating a pheromone inducible HIS3 gene).

[0564] can 1 . . . arginine/canavinine permease.

[0565] ste14::trp1::L gene disruption of STE14, a C-farnesyl

[0566] YS2 . . . methyltransferase (thereby lowering basal signalingthrough the pheromone pathway).

[0567] ste3D1156 . . . endogenous yeast STR, the a factor pheromonereceptor (STE3) was disrupted.

[0568] lys2 . . . defect in 2-aminoapidate reductase, yeast need lysineto grow.

[0569] ura3 . . . defect in orotidine-5′-phosphate decarboxylase, yeastneed uracil to grow

[0570] leu2 . . . defect in b-isopropylmalate dehydrogenase, yeast needleucine to grow.

[0571] trp1 . . . defect in phosphoribosylanthranilate, yeast needtryptophan to grow.

[0572] his3 . . . defect in imidazoleglycerolphosphate dehydrogenase,yeast need histidine to grow.

[0573] Two plasmids were transformed into strain CY7967 byelectroporation: plasmid p5095 (encoding human A₁ adenosine receptor;described above) and plasmid p1584, which is a FUS1-β-galactosidasereporter gene plasmid. Plasmid pl584 was derived from plasmid pRS426(Christianson, T. W. et al. (1992) Gene 110:119-1122). Plasmid pRS426contains a polylinker site at nucleotides 2004-2016. A fusion betweenthe FUS1 promoter and the β-galactosidase gene was inserted at therestriction sites EagI and XhoI to create plasmid p1584. The p1584plasmid is maintained by Trp selection (i.e., growth on medium lackingleucine).

[0574] The resultant strain carrying p5095 and pl584, referred to asCY12660, expresses the human A₁ adenosine receptor. To grow this strainin liquid or on agar plates, minimal media lacking leucine andtryptophan was used. To perform a growth assay on plates (assayingFUS1-HIS3), the plates were at pH 6.8 and contained 0.5-2.5 mM3-amino-1,2,4-triazole and lacked leucine, tryptophan and histidine. Asa control for specificity, a comparison with one or more otheryeast-based seven transmembrane receptor screens was included in allexperiments.

[0575] Construction of Yeast Strains Expressing Human A_(2a) AdenosineReceptor

[0576] In this example, the construction of yeast strains expressing ahuman A_(2a) adenosine receptor functionally integrated into the yeastpheromone system pathway is described.

[0577] I. Expression Vector Construction

[0578] To construct a yeast expression vector for the human A2aadenosine receptor, the human A_(2a) receptor cDNA was obtained from Dr.Phil Murphy (NIH). Upon receipt of this clone, the A_(2a) receptorinsert was sequenced and found to be identical to the published sequence(GenBank accession # S46950). The receptor cDNA was excised from theplasmid by PCR with VENT polymerase and cloned into the plasmid pLPBX,which drives receptor expression by a constitutive PhosphoglycerateKinase (PGK) promoter in yeast. The sequence of the entire insert wasonce again sequenced and found to be identical with the publishedsequence. However, by virtue of the cloning strategy employed there werethree amino acids appended to the carboxy-terminus of the receptor,GlySerVal.

[0579] II. Yeast Strain Construction

[0580] To create a yeast strain expressing the human A_(2a) adenosinereceptor, yeast strain CY8342 was used as the starting parental strain.The genotype of CY8342 is as follows: MATa far1D1442 tbt1-1 lys2 ura3leu2 trpl his3 fus1-HIS3 can1 ste3D1156 gpaD1163 ste14::trp1::LYS2gpa1p-rG_(αs)E10K (or gpa1p-rG(XSD229S or gpalp-rG_(αs)E10K+D229S)

[0581] The genetic markers are as described in Example 1, except for theG-protein variation. For human A_(2a) receptor-expression, yeast strainswere utilized in which the endogenous yeast G protein GPAL had beendeleted and replaced by a mammalian G_(αs). Three rat Gas mutants wereutilized. These variants contain one or two point mutations whichconvert them into proteins which couple efficiently to yeast β_(γ). Theyare identified as G_(αs)E10K (in which the glutamic acid at position tenis replaced with lysine), G_(αs)D229S (in which the aspartic acid atposition 229 is replaced with serine) and G_(αs)E10K+D229S (whichcontains both point mutations).

[0582] Strain CY8342 (carrying one of the three mutant rat G_(αs)proteins) was transformed with either the parental vector pLPBX(Receptor⁻) or with pLPBX-A_(2a) (Receptor⁺). A plasmid with the FUS1promoter fused to β-galactosidase coding sequences (described in above)was added to assess the magnitude of activation of the pheromoneresponse pathway.

[0583] Functional Assay using Yeast Strains Expressing Human A₁Adenosine Receptor

[0584] In this example, the development of a functional screening assayin yeast for modulators of the human A₁ adenosine receptor is described.

[0585] I. Ligands Used in Assay

[0586] Adenosine, a natural agonist for this receptor, as well as twoother synthetic agonists were utilized for development of this assay.Adenosine, reported to have an EC₅₀ of approximately 75 nM, and(-)-N6-(2-phenylisopropyl)-adenosine (PIA) with a reported affinity ofapproximately 50 nM were used in a subset of experiments.5′-N-ethylcarboxamido-adenosine (NECA) was used in all growth assays. Toprevent signaling due to the presence of adenosine in the growth media,adenosine deaminase (4U/ml) was added to all assays.

[0587] II. Biological Response in Yeast

[0588] The ability of the A₁ adenosine receptor to functionally couplein a heterologous yeast system was assessed by introducing the A₁receptor expression vector (p5095, described above) into a series ofyeast strains that expressed different G protein subunits. The majorityof these transformants expressed G_(α) subunits of the G_(αi) or G_(αo)subtype. Additional G_(α) proteins were also tested for the possibleidentification of promiscuous receptor-Gα protein coupling. In variousstrains, a STEl8 or a chimeric STE18-Gγ2 construct was integrated intothe genome of the yeast. The yeast strains harbored a defective HIS3gene and an integrated copy of FUS1-HIS3, thereby allowing for selectionin selective media containing 3-amino-1,2,4-triazole (tested at 0.2, 0.5and 1.0 mM) and lacking histidine. Transformants were isolated andmonolayers were prepared on media containing 3-amino-1,2,4-triazole, 4U/ml adenosine deaminase and lacking histidine. Five microliters ofvarious concentrations of ligand (e.g., NECA at 0, 0.1, 1.0 and 10 mM)was applied. Growth was monitored for 2 days. Ligand-dependent growthresponses were tested in this manner in the various yeast strains. Theresults are summarized in Table 1 below. The symbol (−) indicates thatligand-dependent receptor activation was not detected while (+) denotesligand-dependent response. The term “LIRMA” indicates ligand independentreceptor mediated activation. TABLE 3 Yeast Gγ Strain strain Gα subunitsubunit Variants Result CY1316 GPA₁ STE18 − GPA41-G_(αi1) +GPA41-G_(αi2) + GPA41-G_(αi3) + GPA41-G_(ai2)-G_(αOB) LIRMAGPA41-G_(αSE10K) − GPA41-G_(αSD2295) − CY7967 GPA41-G_(αi3)- STE18 +++integrated CY2120 GPA₁ STE18 sst2Δ + GPA41-G_(αi1) + GPA41-G_(αi2) +GPA41-G_(αi3) + GPA41-G_(αi2)-G_(αOB) LIRMA GPA41-G_(αSE10K) −GPA41-G_(αSD2295) − CY9438 GPA₁ STE18-Gγ2 − GPA41-G_(αi1) +GPA41-G_(αi2) + GPA41-G_(αi3) + GPA41-G_(αi2)-G_(αOB) LIRMAGPA41-G_(αSE10K) − GPA41 -G_(αSD2295) − CY10560 GPA₁-integratedSTE18-Gγ2 sst2Δ ++

[0589] As indicated in Table 3, the most robust signaling was found tooccur in a yeast strain expressing the GPA₁(41)-G_(αi3) chimera.

[0590] III. fus1-LacZ Assay

[0591] To characterize activation of the pheromone response pathway morefully, synthesis of β-galactosidase through fus1LacZ in response toagonist stimulation was measured. To perform the β-galactosidase assay,increasing concentrations of ligand were added to mid-log culture ofhuman A₁ adenosine receptor expressed in a yeast strain co-expressing aSte18-Gγ2 chimera and GPA₄₁-G_(αi3). Transformants were isolated andgrown overnight in the presence of histidine and 4 U/ml adenosinedeaminase. After five hours of incubation with 4 U/ml adenosinedeaminase and ligand, induction of β-galactosidase was measured usingCPRG as the substrate for β-galactoside. 5×10⁵ cells were used perassay.

[0592] The results obtained with NECA stimulation indicated that at aNECA concentration of 10⁻⁸ M approximately 2-fold stimulation ofβ-galactosidase activity was achieved. Moreover, a stimulation index ofapproximately 10-fold was observed at a NECA concentration of 10⁻⁵ M.

[0593] The utility of this assay was extended by validation of theactivity of antagonists on this strain. Two known adenosine antagonist,XAC and DPCPX, were tested for their ability to compete against NECA (at5 mM) for activity in the β-galactosidase assay. In these assays,β-galactosidase induction was measured using FDG as the substrate and1.6×10⁵ cells per assay. The results indicated that both XAC and DPCPXserved as potent antagonists of yeast-expressed A₁ adenosine receptor,with IC₅₀ values of 44 nM and 49 nM, respectively.

[0594] In order to determine if this inhibitory effect was specific tothe A₁ subtype, a series of complementary experiments were performedwith the yeast-based A_(2a) receptor assay (described in Example 4).Results obtained with the A_(2a) yeast-based assay indicated that XACwas a relatively effective A_(2a) receptor antagonist, consistent withpublished reports. In contrast, DPCPX was relatively inert at thisreceptor, as expected from published reports.

[0595] IV. Radioligand Binding

[0596] The A₁ adenosine receptor assay was further characterized bymeasurement of the receptor's radioligand binding parameters.Displacement binding of [³H]CPX by several adenosine receptor referencecompounds, XAC, DPCPX, and CGS, was analyzed using membranes preparedfrom yeast expressing the human Al adenosine receptor. The results withyeast membranes expressing the human A₁ adenosine receptor were comparedto those from yeast membranes expressing the human A_(2a) adenosinereceptor or the human A3 receptor to examine the specificity of binding.To perform the assay, fifty mg of membranes were incubated with 0.4 nM[³H]CPX and increasing concentrations of adenosine receptor ligands.Incubation was in 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 10 mM MgCl₂, 0.25 WBSA and 2 U/ml adenosine deaminase in the presence of proteaseinhibitors for 60 minutes at room temperature. Binding was terminated byaddition of ice-cold 50 mM Tris-HC1, pH 7.4 plus 10 mM MgCl₂, followedby rapid filtration over GF/B filters previously soaked with 0.5%polyethyenimine, using a Packard 96-well harvester. Data were analyzedby nonlinear least square curve fitting procedure using Prism 2.01software. The IC₅₀ values obtained in this experiment are summarized inTable 4, below: TABLE 4 IC₅₀ [nM] Compound hA1R hA2aR hA3R XAC 6.6 11.753.1 DPCPX 8.5 326.4 1307.0 CGS-15943 13.1 15.8 55.5 NECA 215.5 294.934.9 R-PIA 67.6 678.1 23.6 IB-MECA 727.7 859.4 3.1 Alloxozine 1072.01934.0 8216.0

[0597] These data indicate that the reference compounds have affinitiesconsistent with those reported in the literature. The data furtherindicate that the yeast-based assays are of sufficient sensitivity todiscriminate receptor subtype specificity.

[0598] Functional Assay using Yeast Strains Expressing Human A_(2a)Adenosine Receptor

[0599] In this example, the development of a functional screening assayin yeast for modulators of the human A₁ adenosine receptor is described.

[0600] I. Ligands Used in Assay

[0601] The natural ligand adenosine, as well as other thoroughlycharacterized and commercially available ligands were used for study ofthe human A_(2a) receptor functionally expressed in yeast. Three ligandshave been used in the establishment of this assay. They include: LigandReported K_(i) Function Adenosine 500 nM agonist5′-N-ethylcarboxamidoadenosine 10-15 nM agonist (NECA) (−)-NG-(2-100-125 nM agonist phenylisopropyl)-adenosine (PIA)

[0602] To prevent signaling due to the presence of adenosine in thegrowth media, adenosine deaminase (4U/ml) was added to all assays.

[0603] II. Biological Response in Yeast

[0604] A_(2a) receptor agonists were tested for the capacity tostimulate the pheromone response pathway in yeast transformed with theA_(2a) receptor expression plasmid and expressing either G_(αs)E10K,G_(αs)D229S or G_(αs)E10K+D229S. The ability of ligand to stimulate thepheromone response pathway in a receptor dependent manner was indicatedby an alteration in the yeast phenotype. Receptor activation modifiedthe phenotype from histidine auxotrophy to histidine prototrophy(activation of fus1-HIS3). Three independent transformants were isolatedand grown overnight in the presence of histidine. Cells were washed toremove histidine and diluted to 2×10⁶ cells/ml. 5 μl of eachtransformant was spotted onto nonselective media (including histidine)or selective media (1 mM AT) in the absence or presence of 4 U/mladenosine deaminase. Plates were grown at 30° C. for 24 hours. In thepresence of histidine both Receptor⁺ (R⁺) and Receptor⁻ (R⁻) strainswere capable of growth. However, in the absence of histidine only R⁺cells grew. Since no ligand had been added to these plates twoexplanations were possible for this result. One possible interpretationwas that the receptor bearing yeast were at a growth advantage due toLigand Independent Receptor Mediated Activation (LIRMA). Alternativelythe yeast could have been synthesizing the ligand adenosine. Todistinguish between these two possibilities, an enzyme which degradesthe ligand, adenosine deaminase (ADA), was added to the growing yeastand plates. In the presence of adenosine deaminase R⁺ cells no longergrew in the absence of histidine, indicating that the yeast were indeedsynthesizing ligand.

[0605] This interpretation was confirmed by an A_(2a) growth assay inliquid. In this experiment R⁺ yeast (a G_(αs)E10K strain expressing theA_(2a) receptor) were inoculated at three densities (1×10⁶ cell/ml;3×10⁵ cells/ml; or 1×10⁵ cells/ml) in the presence or absence ofadenosine deaminase (4 U/ml). The stringency of the assay was enhancedwith increasing concentrations (0, 0.1, 0.2 or 0.4 mM)of3-amino-1,2,4-triazole (AT), a competitive antagonist ofimidazoleglycerol-P dehydratase, the protein product of the HIS3 gene.In the presence of adenosine deaminase and 3-amino-1,2,4-triazole yeastgrew less vigorously. However in the absence of 3-amino-1,2,4-triazole,adenosine deaminase had little effect. Thus adenosine deaminase itselfhad no direct effect upon the pheromone response pathway.

[0606] An alternative approach to measuring growth and one that can beminiaturized for high throughput screening is an A_(2a) receptor ligandspot assay. A G_(αs)ElOK strain expressing the A_(2a) receptor (A2aR+)or lacking the receptor (R-) was grown overnight in the presence ofhistidine and 4 U/ml adenosine deaminase. Cells were washed to removehistidine and diluted to 5×10⁶ cells/ml. 1×10⁶ cells were spread ontoselective plates containing 4 U/ml adenosine deaminase and 0.5 or 1.0 mM3-amino-1,2,4-triazole (AT) and allowed to dry for 1 hour. 5 μl of thefollowing reagents were applied to the monolayer: 10 mM adenosine, 38.7mM histidine, dimethylsulfoxide (DMSO), 10 EM PIA or 10 mM NECA. Cellswere grown 24 hours at 30° C. The results showed that cells withoutreceptor could only grow when histidine was added to the media. Incontrast, R⁺ cells only grew in areas where the A_(2a) receptor ligandsPIA and NECA had been spotted. Since the plates contained adenosinedeaminase, the lack of growth where adenosine had been spotted confirmedthat adenosine deaminase was active.

[0607] III. fus1 LacZ Assay

[0608] To quantitate activation of the yeast mating pathway, synthesisof β-galactosidase through fus1LacZ was measured. Yeast strainsexpressing G_(αs)ElOK, G_(αs)D229S or G_(αs)E10K+D229S were transformedwith a plasmid encoding the human A_(2a) receptor (R+) or with a plasmidlacking the receptor (R−). Transformants were isolated and grownovernight in the presence of histidine and 4 U/ml adenosine deaminase.1×10⁷ cells were diluted to 1×10⁶ cells/ml and exposed to increasingconcentrations of NECA for 4 hours, followed by determination of theβ-galactosidase activity in the cells. The results demonstrated thatessentially no β-galactosidase activity was detected in R− strains,whereas increasing amounts of β-galactosidase activity were detected inR+ strains expressing either G_(αs)E10K, G_(αs)D229S or G_(αs)E10K+D229Sas the concentration of NECA increased, indicating a dose dependentincrease in units of β-galactosidase detected in response to exposure toincreased ligand concentration. This dose dependency was only observedin cells expressing the A_(2a) receptor. Furthermore the most potentG_(αs) construct for the A_(2a) receptor was G_(60 s)E10K. TheG_(αs)D229S construct was the second-most potent G_(αs) construct forthe A_(2a) receptor, while the G_(αs)E10K+D229S construct was the leastpotent of the three G_(αs) constructs tested, although even theG_(αs)E10K+D229S construct stimulated readily detectable amounts ofβ-galactosidase activity.

[0609] For a further description of the assays identified, see U.S.application Ser. No. 09/088985, entitled “Functional Expression ofAdenosine Receptors in Yeast”, filed Jun. 2, 1998 (Attorney Docket No.CPI-093), the entire contents of which are hereby incorporated herein byreference.

[0610] Pharmacological Characterization of the Human Adenosine ReceptorSubtypes

[0611] Material and Methods

[0612] Materials. [³H] -DPCPX [Cyclopentyl-1,3-dipropylxantine,8-[dipropyl-2,3-³H(N)] (120.0 Ci/mmol); [³H]-CGS 21680, [carboxyethyl-³H(N)] (30 Ci/mmol) and [¹²⁵I] -AB-MECA([¹²⁵I]-4-Aminobenzyl-5′-N-Methylcarboxamideoadenosine) (2,200 Ci/mmol)were purchased from New England Nuclear (Boston, Mass.). XAC (Xantineamine congener); NECA (5′-N-Ethylcarboxamidoadenosine); and IB-MECA fromResearch Biochemicals International (RBI, Natick, Mass.). The AdenosineDeaminase and Complete protease inhibitor cocktail tablets werepurchased from Boehringer Mannheim Corp. (Indianapolis, Ind.). Membranesfrom HEK-293 cells stably expressing the human Adenosine 2a [RB-HA2a];Adenosine 2b [RB-HA2b] or Adenosine 3 [RB-HA3] receptor subtypes,respectively were purchased from Receptor Biology (Beltsville, Md.).Cell culture reagents were from Life Technologies (Grand Island, N.Y.)except for serum that was from Hyclone (Logan, Utah).

[0613] Yeast strains: Saccharomyces cerevisiae strains CY12660[far1*1442 tbt1-1 fus1-HIS3 can1 ste14::trp1::LYS2 ste3*1156gpa1(41)-Gαi3 lys2 ura3 leu2 trp1: his3; LEU2PGKp-Mfα1Leader-hAlR-PHO5term 2mu-orig REP3 Ampr] and CY8362[gpa1p-rGαsE10K far1*1442 tbt1-1 fus1-HIS3 can1 ste14::trp1: LYS2ste3*1156 lys2 ura3 leu2 trp1 his3; LEU2 PGKp-hA2aR 2mu-ori REP3 Ampr]were developed as described above.

[0614] Yeast culture: Transformed yeast were grown in Leu-Trp [LT] media(pH 5.4) supplemented with 2% glucose. For the preparation of membranes250 ml of LT medium were inoculated with start titer of 1-2×10⁶ cells/mlfrom a 30 ml overnight culture and incubated at 30° C. under permanentoxygenation by rotation. After 16 h growth the cells were harvested bycentrifugation and membranes were prepared as described below.

[0615] Mammalian Tissue Culture: The HEK-293 cells stably expressedhuman Adenosine 2a receptor subtype (Cadus clone #5) were grown inDulbeco's minimal essential media (DMEM) supplemented with 10% fetalbovine serum and 1× penicillin/streptomycin under selective pressureusing 500 mg/ml G418 antibiotic, at 37° C. in a humidified 5% CO₂atmosphere.

[0616] Yeast Cell Membrane Preparations: 250 ml cultures were harvestedafter overnight incubation by centrifugation at 2,000×g in a SorvallRT6000 centrifuge. Cells were washed in ice-cold water, centrifuged at4° C. and the pellet was resuspended in 10 ml ice-cold lysis buffer [5mM Tris-HCl, pH 7.5; 5 mM EDTA; and 5 mM EGTA] supplemented withProtease inhibitor cocktail tablets (1 tablet per 25 ml buffer) Glassbeads (17 g; Mesh 400-600; Sigma) were added to the suspension and thecells were broken by vigorous vortexing at 4° C. for 5 min. Thehomogenate was diluted with additional 30 ml lysis buffer plus proteaseinhibitors and centrifuged at 3,000×g for 5 min. Subsequently themembranes were peleted at 36,000×g (Sorvall RC5B, type SS34 rotor) for45 min. The resulting membrane pellet was resuspended in 5 ml membranebuffer [50 mM Tris-HCl, pH 7.5; 0.6 mM EDTA; and 5 mM MgCl₂]supplemented with Protease inhibitor cocktail tablets (1 tablet per 50ml buffer) and stored at −80° C. for further experiments.

[0617] Mammalian Cell Membrane Preparations: HEK-293 cell membranes wereprepared as described previously (Duzic E et al.: J. Biol. Chem., 267,9844-9851, 1992) Briefly, cells were washed with PBS and harvested witha rubber policeman. Cells were pelted at 4° C. 200×g in a Sorvall RT6000centrifuge. The pellet was resuspended in 5 ml/dish of lysis buffer at4° C. (5 mM Tris-HCl, pH 7.5; 5 mM EDTA; 5 mM EGTA; 0.1 mMPhenylmethylsulfonyl fluoride, 10 mg/ml pepstatin A; and 10 mg/mlaprotinin) and homogenized in a Dounce homogenizer. The cell lysate wasthen centrifuged at 36,000×g (Sorvall RCSB, type SS34 rotor) for 45 minand the pellet resuspended in 5 ml membrane buffer [50 mM Tris-HCl, pH7.5; 0.6 mM EDTA; 5 mM MgCl₂; 0.1 mM Phenylmethylsulfonyl fluoride, 10mg/ml pepstatin A; and 10 mg/ml aprotinin) and stored at −80° C. forfurther experiments.

[0618] The Bio-Rad protein assay kits, based on the Bradford dye-bindingprocedure, (Bradford, M.: Anal. Biochem. 72:248 (1976)) were used todetermine total protein concentration in yeast and mammalian membranes.

[0619] Adenosine 1 receptor subtype saturation and competitionradioligand binding: Saturation and competition binding on membranesfrom yeast cell transformed with human A₁ receptor subtype were carriedout using antagonist [³H] DPCPX as a radioactive ligand. Membranes wasdiluted in binding buffer [50 mM Tris-HCl, pH 7.4; containing 10 mMMgCl₂; 1.0 mM EDTA; 0.25% BSA; 2 U/ml adenosine deaminase and 1 proteaseinhibitor cocktail tablet/50 ml] at concentrations of 1.0 mg/ml.

[0620] In saturation binding membranes (50 μg/well) were incubate withincreasing concentrations of [³H] DPCPX (0.05-25 nM) in a final volumeof 100 μl of binding buffer at 25° C. for 1 hr in the absence andpresence of 10 μM unlabeled XAC in a 96-well microtiter plate.

[0621] In competition binding membranes (50 μg/well) were incubate with[³H] DPCPX (1.0 nM) in a final volume of 100 ml of binding buffer at 25°C. for 1 hr in the absence and presence of 10 μM unlabeled XAC orincreasing concentrations of competing compounds in a 96-well microtiterplate.

[0622] Adenosine 2a receptor subtype competition radioligand binding:Competition binding on membranes from HEK293 cell stably expressing thehuman A_(2a) receptor subtype were carried out using agonist [³H]CGS-21680 as a radioactive ligand. Membranes was diluted in bindingbuffer [50 mM Tris-HCl, pH 7.4; containing 10 mM MgCl₂; 1.0 mM EDTA;0.25% BSA; 2 U/ml adenosine deaminase and 1 protease inhibitor cocktailtablet/50 ml] at concentrations of 0.2 mg/ml. Membranes (10 μg/well)were incubate with [³H] CGS-21680 (100 nM) in a final volume of 100 mlof binding buffer at 25° C. for 1 hr in the absence and presence of 50μM unlabeled NECA or increasing concentrations of competing compounds ina 96-well microtiter plate.

[0623] Adenosine 3 receptor competition radioligand binding: Competitionbinding on membranes from HEK293 cell stably expressing the human A₃receptor subtype were carried out using agonist [¹²⁵I] AB-MECA as aradioactive ligand. Membranes was diluted in binding buffer [50 mMTris-HCl, pH 7.4; containing 10 mM MgCl₂; 1.0 mM EDTA; 0.25% BSA; 2 U/mladenosine deaminase and 1 protease inhibitor cocktail tablet/50 ml] atconcentrations of 0.2 mg/ml. Membranes (10 μg/well) were incubate with[¹²⁵ I] AB-MECA (0.75 nM) in a final volume of 100 μl of binding bufferat 25° C. for 1 hr in the absence and presence of 10 μM unlabeledIB-MECA or increasing concentrations of competing compounds in a 96-wellmicrotiter plate.

[0624] At the end of the incubation, the A₁, A_(2a) and A₃ receptorsubtypes radioligand binding assays was terminated by the addition ofice-cold 50 mM Tris-HCl (pH 7.4) buffer supplemented with 10 mM MgCl₂,followed by rapid filtration over glass fiber filters (96-well GF/BUniFilters, Packard) previously presoaked in 0.5% polyethylenimine in aFiltermate 196 cell harvester (Packard). The filter plates were driedcoated with 50 μl /well scintillation fluid (MicroScint-20, Packard) andcounted in a TopCount (Packard). Assays were performed in triplicate.Non-specific binding was 5.6±0.5%, 10.8±1.4% and 15.1±2.6% of the totalbinding in a A₁R, A_(2a)R and A₃R binding assay, respectively.

[0625] Adenosine 2b receptor subtype competition radioligand binding:Competition binding on membranes from HEK293 cell stably expressing thehuman A_(2b) receptor subtype were carried out using A₁ receptorantagonist [³H] DPCPX as a radioactive ligand. Membranes was diluted inbinding buffer [10 mM Hepes-KOH, pH 7.4; containing 1.0 mM EDTA; 0.1 mMBenzamidine and 2 U/ml adenosine deaminase] at concentrations of 0.3mg/ml. Membranes (15 μg/well) were incubate with [³H] DPCPX (15 nM) in afinal volume of 100 μl of binding buffer at 25° C. for 1 hr in theabsence and presence of 10 μM unlabeled XAC or increasing concentrationsof competing compounds in a 96-well microtiter plate. At the end of theincubation, the assay was terminated by the addition of ice-cold 10 mMHepes-KOH (pH 7.4) buffer followed by rapid filtration over glass fiberfilters (96-well GF/C UniFilters, Packard) previously presoaked in 0.5%polyethylenimine in a Filtermate 196 cell harvester (Packard). Thefilter plates were dried coated with 50 μl/well scintillation fluid(MicroScint-20, Packard) and counted in a TopCount (Packard). Assayswere performed in triplicate. Non-specific binding was 14.3±2.3% of thetotal binding.

[0626] Specific binding of [³H] DPCPX; [³H] CGS-21680 and [1251] AB-MECAwas defined as the difference between the total binding and non-specificbinding. Percent inhibition of the compounds was calculated againsttotal binding. Competition data were analyzed by iterative curve fittingto a one site model, and K_(I) values were calculated from IC5₀ values(Cheng and Prusof, Biochem. Pharmacol. 22, 3099-3109, 1973) using theGraphPad Prizm 2.01 software.

[0627] Results

[0628] A primary function of certain cell surface receptors is torecognize appropriate ligands. Accordingly, we determined ligand bindingaffinities to establish the functional integrity of the Adenosine 1receptor subtype expressed in yeast. Crude membranes prepared fromSaccharomyces cerevisiae transformed with human Adenosine 1 receptorsubtype construct exhibited specific saturable binding of [³H] DPCPXwith a K_(D) of 4.0±0.19 nM. The K_(D) and B_(max) value were calculatedfrom the saturation isotherm and Scatchard transformation of the dataindicated a single class of binding sites. The densities of adenosinebinding sites in the yeast membrane preparations were estimated to716.8±43.4 fmol/mg membrane protein.

[0629] The pharmacological subtype characteristics of the recombinantyeast cells transformed with human A₁ receptor subtype were investigatedwith subtype selective adenosine ligands (XAC, DPCPX; CGS-15943;Compound 600; Compound 1002; NECA, (R)-PIA; IB-MECA and Alloxazine) thatcompeted with [³H] DPCPX in the expected rank order. Displacement curvesrecorded with these compounds show the typical steepness with all theligands, and the data for each of the ligands could be modeled by aone-site fit. The apparent dissociation constants estimated for theindividual compound from the curves (Table 5) are consistent with valuepublished for the receptor obtained from other sources. TABLE 5 Kivalues for membranes from yeast cells transformed with human A₁ receptorsubtype Ligands K_(I) (nM) XAC 5.5 DPCPX 7.1 CGS-1594 10.8 NECA 179.6(R)-PIA 56.3 IB-MECA 606.5 Alloxazine 894.1 Compound 600 13.9 Compound1002 9.8

[0630] Tables 6 through 12 demonstrate the efficacy and structureactivity profiles of deazapurines of the invention. Tables 13 and 14demonstrate selectivity can be achieved for human adenosine receptorsites by modulation of the functionality about the deazapurinestructure. Table 14 also demonstrates the surprising discovery that thecompounds set forth therein have subnanomolar activity and higherselectivity for the A_(2b) receptor as compared to the compounds inTable 13. TABLE 6 Effect of N₆-Substituent

A1 Com- Binding Yeast pound R Ki (nM) IC50 (nM) 600

13.9 97.2 601

1423 >10.000 602

483.5 >10.000 603

196.6 4442.0 604

>10.000 >10000 605

>10000 >10000 606

297.9 >10000 607

309.7 >10000 608

29.1 609

193.9 610

411.5 611

785.6 >10000 612

64.8 613

6726.0 614

32.1 615

816.9 2577.0 616

34.3

[0631] TABLE 7 Effect of C₂-Substituent

A1 Binding Yeast Compound R Ki (nM) IC50 (nM) 700

604.5 >10000 701

157.7 763.1 702

198.5 2782.5 703

443.6 >10000 704

61.1 297.0 705

30.1 194.7 706

19.9 707

62.8 708

2145 709

48.7

[0632] TABLE 8 Effect of Pyrrole Ring Substituent

A1 Yeast Binding IC50 Compound R R′ R″ R′″ Ki (nM) (nM) 800

Me Me Me 3311 >10000 801

H Me H 22.3 148.3 802

H H Me 8.9 803

Me Me 2210 >10000 804

Me Me 863.1 805

Me Me 4512 806

Me Me 8451 807

Me Me 35.3

[0633] TABLE 9

A1 Yeast Binding IC50 Compound R Ki (nM) (nM) 900

863.1 901

4512 902

8451 903

35.3

[0634] TABLE 10 Effect of N₆-Substituent

A1 Binding Yeast Compound R Ki (nM) IC50 (nM) 1000

1789 >10000 1001

54.4 1865 1002

9.8 82.8 1003

26.7 195.7 1004

32.8 545.8 1005

147.5 3972 1006

151.7 2918 1007

692.5 >10000 1008

93.1 3217 1009

475.3 >10000 1010

674.9 9376.0 1011

121.9 2067.5 1012

233.9 3462 1013

270.1 3009.5 1014

384.9 2005 1015

179.3 3712 1016

176.1 5054

[0635] TABLE 11 Effect of N₆-Substituent

A1 Com- Binding Yeast pound R Ki (nM) IC50 (nM) 1100

9.8 115.4 1101

53.9 551.0 1102

10.3 101.3 1103

71.1 3217 1104

6.5 58.7 1105

105.4 472.1 1106

27.8 162.4 1107

126.5 1297.0 1108

2.3 1109

9.0 1110

17.3 1111

2.5 1112

213

[0636] TABLE 12 “Retro-Amide” Analogues

A1 Com- Binding Yeast pound R Ki (nM) IC50 (nM) 1200

16.5 189.4 1201

7.4 45.7 1202

95.8 3345.0 1203

529.1 4040.0 1204

1060.0 >10000 1205

1272 >10000 1206

50.8 4028 1207

48.5 701.5

[0637] TABLE 13 Profile of Selective Adenosine Antagonists

Binding Ki (nM) Compound R A1 A2a A2b A3 1300

9.8-25.1 18.0-48.6 80.3 513.0 1301

27.8 50.7 84.6 429.8 1302

20.2 75.6 20.1 4.3 1303

17.4 111.3 120.6 44.6 1304

13.9-30.9 933.7 138.0 21.5 1305

46.6 730.9 30% 9.9  1306¹

16.4 766.3 168.3 71.7 1307

29.1 190.6 1143.0 3.1 1308

180 230 670 1.0 1309

40 109 109 0.3 1310

255 76% 275 ≦2.6 1311

531 981 736 5.3 1312

443 2965 375 ≦6.2  1313³

30% 65% 515 24 1314

87 204 30 0.02 1315

75.000 720.000 3.400 507 1316

333 710.000 710.000 97 1317

710.000 710.000 720.000 369 1318

3.7 ± 0.5 630 ± 56.4 2307 ± 926 630 ± 76 1319

1.8 206 802 270 1320

8.0 531 530 419  1321⁴

8.0 131 1.031 54%⁸

[0638] TABLE 14 Profile of Selective A_(2o) Antagonists

Binding Data K₁ (nM) Compound XR₁ R₂ A₁ A_(2a) A_(2B) A₃ 1400 —O—Ph Me41.7 21 10.3 14.6 1401 —O—Ph(p)F Me 33 58 8.8 18 1402 —O—Ph(p)Cl Me 825591 22 60 1403 —N-pyridin- Me 60 41 18 48 2-one 1404 —NH—Ph Me 49 31 4.657

[0639] TABLE 15 Adenosine A_(2a) Receptor Selective Compounds RelativeRelative Relative Compound Structure Ki-A1 Ki-A2a Ki-A2b Ki-A3 1600

* 1601

* 1602

* 1603

* 1604

* 1605

* 1606

* 1607

* 1608

*

[0640] This invention provides a compound having the structure:

[0641] This invention also provides a compound having the structure:

[0642] In a further embodiment the invention provides a method fortreating a disease associated with A_(2a) adenosine receptor in asubject, comprising administering to the subject a therapeuticallyeffective amount of compounds 1609 or 1610.

[0643] The invention also provides the above method, wherein the subjectis a mammal.

[0644] The invention further provides the above method, wherein themammal is a human.

[0645] The invention also provides the method for treating a diseaseassociated with A_(2a) adenosine receptor in a subject, wherein theA_(2a) adenosine receptor is associated with locomotor activity,vasodilation, platelet inhibition, neutrophil superoxide generation,cognitive disorder, senile dementia, or Parkinson's disease.

[0646] The invention provides the above method, wherein the compoundtreats the diseases by stimulating adenylate cyclase.

[0647] The invention also provides a water-soluble prodrug of thecompound 1609 or 1610, wherein the water-soluble prodrug is metabolizedin vivo to an active drug to selectively inhibit an A_(2a) adenosinereceptor.

[0648] The invention also provides a water-soluble prodrug of thecompound 1609 or 1610, wherein the prodrug is metabolized in vivo byesterase catalyzed hydrolysis.

[0649] The invention also provides a pharmaceutical compositioncomprising the water-soluble prodrug of the compound 1609 or 1610, and apharmaceutically acceptable carrier.

[0650] The invention also provides a method for inhibiting the activityof an A_(2a) adenosine receptor in a cell, which comprises contactingthe cell with compound 1609 or 1610.

[0651] The invention also provides a method for inhibiting the activityof an A_(2a) adenosine receptor in a cell, which comprises contactingthe cell with compound 1609 or 1610, wherein the compound is anantagonist of said A_(2a) adenosine receptor.

[0652] The invention also provides the above method, wherein the cell isa human cell.

[0653] The invention also provides the above method, wherein the cell isa human cell and the compound is an antagonist of A_(2a) adenosinereceptors.

[0654] The invention also provides a pharmaceutical compositioncomprising a therapeutically effective amount of the compound 1609 or1610 and a pharmaceutically acceptable carrier.

[0655] The invention also provides the above pharmaceutical composition,wherein the therapeutically effective amount is effective to treatParkinson's disease and diseases associated with locomotor activity,vasodilation, platelet inhibition, neutrophil superoxide generation,cognitive disorder, or senile dementia.

[0656] The invention also provides the above pharmaceutical composition,wherein the pharmaceutical composition is an ophthalmic formulation.

[0657] The invention also provides the above pharmaceutical composition,wherein the pharmaceutical composition is an periocular, retrobulbar orintraocular injection formulation.

[0658] The invention also provides the above pharmaceutical composition,wherein the pharmaceutical composition is a systemic formulation.

[0659] The invention also provides the above pharmaceutical composition,wherein the pharmaceutical composition is a surgical irrigatingsolution.

[0660] The invention also provides a combination therapy for Parkinson'sdisease, comprising the compounds 1609 or 1610, and any of the dopamineenhancers.

[0661] The invention also provides a combination therapy for cancer,comprising the compound 1609 or 1610, and any of the cytotoxic agents.

[0662] The invention also provides a combination therapy for glaucoma,comprising the compound 1609 or 1610, and a prostaglandin agonist, amuscrinic agonist, or a β-2 antagonist.

[0663] The invention also provides a packaged pharmaceutical compositionfor treating a disease associated with A_(2a) adenosine receptor in asubject, comprising:

[0664] (a) a container holding a therapeutically effective amount of thecompound 1609 or 1610; and

[0665] (b) instructions for using the compound for treating said diseasein a subject.

[0666] Exemplification

EXAMPLE 20

[0667] Synthesis of1-(6-Phenyl-2-pyridin-4-yl-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-pyrrolidine-2-carboxylicacid amide (1609).

[0668] Compound 1609 was synthesized by reacting L-prolinamide with theappropriate chloride intermediate described in synthesis scheme II onpage 76 to obtain:

[0669]¹H-NMR (d₆-DMSO) d 1.95-2.15 (m, 4H), 4.00 (brs, 1H), 4.15 (brs,1H), 4.72 (brs, 1H), 6.90 (brs, 1H), 7.19 (brs, 1H), 7.30 (t, 1H, J=7.0Hz), 7.44 (t, 2H, J=7.0 Hz), 7.59 (s, 1H), 7.92 (brs, 2H), 8.26 (d,2H,J=6.2 Hz), 8.65 (d, 2H, J=6.2 Hz); MS (ES): 384.9 (M⁺+1); Mpt=280-316°C. (decomp.).

EXAMPLE 21

[0670] Synthesis of1-[6-(3-Methoxy-phenyl)-2-pyridin-4-yl-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-pyrrolidine-2-carboxylicacid amide (1610).

[0671] Compound 1610 was synthesized by reacting L-prolinamide with theappropriate chloride intermediate described in synthesis scheme II onpage 76 to obtain:

[0672]¹H-NMR (d₆-DMSO) d 2.07(m,4H), 3.85(s,3H), 4.02(m,1H), 4.17(m,1H),4.75(m,1H), 6.89(m,1H), 7.00(s,1H), 7.23(s,1H), 7.35(t,1H,J=8.2 Hz),7.53(s,2H), 7.60(s,1H), 8.28(d,2H,J=5.8 Hz), 8.67(d,2H,J=5.8 Hz),12.37(s,1H); MS (ES): 415.0 (M⁺+1).

[0673] Activity of Compounds

[0674] Adenosine 2a (A_(2a)) receptor subtype competition radio ligandbinding were carried out for compounds 1609 and 1610 as described hereinand inter alia, on pages 146 of this specification. Compounds 1609 and1610 were found have A_(2a) receptor binding affinity and selectivity.

[0675] Incorporation by Reference

[0676] All patents, published patent applications and other referencesdisclosed herein are hereby expressly incorporated herein by reference.

[0677] Equivalents

[0678] Those skilled in the art will recognize, or be able to ascertain,using no more than routine experimentation, many equivalents to specificembodiments of the invention described specifically herein. Suchequivalents are intended to be encompassed in the scope of the followingclaims.

What is claimed is:
 1. A compound having the structure:

wherein NR₁R₂ is a substituted or unsubstituted 4-8 membered ring;wherein Ar is a substituted or unsubstituted four to six membered ring;wherein R₄ is H, alkyl, substituted alkyl, aryl, arylalkyl, amino,substituted aryl, wherein said substituted alkyl is —C(R₈) (R₉)XR₆,wherein X is O, S, or NR₇, wherein R₈ and R₉ are each independently H oralkyl, wherein R₆ and R₇ are each independently alkyl or cycloalkyl, orR₆, R₇ and the nitrogen together form a substituted or unsubstitutedring of between 4 and 7 members. wherein R₅ is H, alkyl, substitutedalkyl, or cycloalkyl; with the proviso that NR₁R₂ is not 3-acetamidopiperadino, 3-hydroxy pyrrolidino, 3-methyloxy carbonylmethylpyrrolidino, or 3-aminocarbonylmethyl pyrrolidino; with the proviso thatNR₁R₂ is 4-hydroxymethyl piperadino only when Ar is 4-pyridyl.
 2. Thecompound of claim 1, wherein Ar is phenyl, pyrrole, thiophene, furan,thiazole, pyridine.
 3. The compound of claim 1, having the structure:

wherein m is 0, 1, 2, or 3; wherein R_(A) and R_(B) are eachindependently be H, —OH, —CH₂OH, —CH₂CH₂OH, —C(═O)NH₂, a heteroatom, or—C(═O)NR₃R₃′; wherein R₃ is aryl, substituted aryl, or heteroaryl;wherein R₃′ is alkyl, or XR₃″, wherein X is O, or N and R″ issubstituted alkyl or aryl.
 4. The compound of claim 1, having thestructure:

wherein m is 0, 1, 2, or 3; wherein Y is O, S, or NR, wherein R is R_(A)or R_(B); wherein R_(A) and R_(B) are each independently be H, —OH,—CH₂OH, —CH₂CH₂OH, —C(═O)NNH₂, a heteroatom, or —C(═O)NR₃R₃ ¹ ; whereinR₃ is aryl, substituted aryl, or heteroaryl; wherein R₃′ is alkyl, orXR₃″, wherein X is O, or N and R″ is substituted alkyl or aryl.
 5. Thecompound of claim 1, wherein R₁R₂N is (D)-2-aminocarbonyl pyrrolidino,(D)-2-hydroxymethyl pyrrolidino, (D)-2-hydroxymethyl- trans-4-hydroxypyrrolidino, piperazino, or 3-hydroxymethyl piperadino.
 6. The compoundof claim 1, having the structure:


7. The compound of claim 1, having the structure:


8. The compound of claim 1, having the structure:


9. The compound of claim 1, having the structure:


10. The compound of claim 1, having the structure:


11. The compound of claim 1, having the structure:


12. The compound of claim 1, having the structure:


13. The compound of claim 1, having the structure:


14. The compound of claim 11, having the structure:


15. The compound of claim 11, having the structure:


16. A compound having the structure(V):

wherein R is H, or methyl.
 17. The compound of claim 16, having thestructure:


18. The compound of claim 16, having the structure:


19. A method for treating a disease associated with A_(2a) adenosinereceptor in a subject, comprising administering to the subject atherapeutically effective amount of a compound of claims 1, or
 16. 20.The method of claim 19, wherein the subject is a mammal.
 21. The methodof claim 20, wherein the mammal is a human.
 22. The method of claim 21,wherein said A_(2a) adenosine receptor is associated with locomotoractivity, vasodilation, platelet inhibition, neutrophil superoxidegeneration, cognitive disorder, senile dementia, or Parkinson's disease.23. The method of claim 19, wherein the compound treats said diseases bystimulating adenylate cyclase.
 24. A water-soluble prodrug of thecompound of claims 1, or 16, wherein said water-soluble prodrug that ismetabolized in vito to an active drug which selectively inhibit A_(2a)adenosine receptor.
 25. The prodrug of claim 24, wherein said prodrug ismetabolized in vivo by esterase catalyzed hydrolysis.
 26. Apharmaceutical composition comprising the prodrug of claim 24 and apharmaceutically acceptable carrier.
 27. A method for inhibiting theactivity of an A_(2a) adenosine receptor in a cell, which comprisescontacting said cell with a compound of claims 1, or
 16. 28. The methodof claim 27, wherein the compound is an antagonist of said A_(2a)adenosine receptor.
 29. The method of claim 28, wherein the cell is ahuman cell.
 30. The method of claim 28, wherein the compound is anantagonist of A_(2a) adenosine receptors.
 31. A pharmaceuticalcomposition comprising a therapeutically effective amount of thecompound of claims 1, or 16 and a pharmaceutically acceptable carrier.32. The pharmaceutical composition of claim 31, wherein saidtherapeutically effective amount is effective to treat Parkinson'sdisease and diseases associated with locomotor activity, vasodilation,platelet inhibition, neutrophil superoxide generation, cognitivedisorder, or senile dementia.
 33. The pharmaceutical composition ofclaim 31, wherein said pharmaceutical composition is an ophthalmicformulation.
 34. The pharmaceutical composition of claim 31, whereinsaid pharmaceutical composition is an periocular, retrobulbar orintraocular injection formulation.
 35. The pharmaceutical composition ofclaim 31, wherein said pharmaceutical composition is a systemicformulation.
 36. The pharmaceutical composition of claim 31, whereinsaid pharmaceutical composition is a surgical irrigating solution.
 37. Acombination therapy for Parkinson's disease, comprising the compounds ofclaims 1 or 16, and any of the dopamine enhancers.
 38. A combinationaltherapy for cancer, comprising the compound of claims 1 or 16, and anyof the cytotoxic agents.
 39. A combinational therapy for glaucoma,comprising the compound of claims 1 or 16, and a prostaglandin agonist,a muscrinic agonist, or a β-2 antagonist.
 40. A packaged pharmaceuticalcomposition for treating a disease associated with A_(2a) adenosinereceptor in a subject, comprising: (a) a container holding atherapeutically effective amount of the compound of claims 1, or 16; and(b) instructions for using said compound for treating said disease in asubject.
 41. A method of preparing the compound of claim 1, comprisingthe steps of a)

wherein P is a removable protecting group; b) treating the product ofstep a) under cyclization conditions to provide

c) treating the product of step b) under suitable conditions to provide

d) treating the chlorinated product of step c) with NR₁R₂ to provide

wherein NR₁R₂ is a substituted or unsubstituted 4-8 membered ring;wherein Ar is a substituted or unsubstituted four to six membered ring,wherein R₄ is H, alkyl, substituted alkyl, aryl, arylalkyl, amino,substituted aryl, wherein said substituted alkyl is —C(R₈) (R₉) XR₆,wherein X is O, S, or NR₇, wherein R₈ and R₉ are each independently H oralkyl, wherein R₆ and R₇ are each independently alkyl or cycloalkyl, orR₆, R₇ and the nitrogen together form a substituted or unsubstitutedring of between 4 and 7 members. wherein R₅ is H, alkyl, substitutedalkyl, or cycloalkyl; with the proviso that NR₁R₂ is not 3-acetamidopiperadino, 3-hydroxy pyrrolidino, 3-methyloxy carbonylmethylpyrrolidino, 3-aminocarbonylmethyl pyrrolidino, or 3-hydroxymethylpiperadino;
 42. A method of preparing the compound of claim 16,comprising the steps of a)

wherein P is a removable protecting group; b) treating the product ofstep a) under cyclization conditions to provide

c) treating the product of step b) under suitable conditions to provide

d) treating the chlorinated product of step c) first with dimethylamineand formaldehyde, then with N-methyl benzylamine and finally with NH₂R₁to provide

wherein R₁ is acetomido ethyl; wherein Ar is 4-pyridyl; wherein R is H,or methyl; wherein R₅ is N-methyl-N-benzyl aminomethyl.